Transposition-mediated identification of specific binding or functional proteins

ABSTRACT

The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the discovery and optimization of polypeptides having desired binding specificity and/or functionality, including antigen-binding molecules such as antibodies and fragments thereof, for desired functional and/or binding phenotypes. The novel method is based on transposable constructs and diverse DNA libraries cloned into transposable vectors and their transfection into host cells by concomitant transient expression of a functional transposase enzyme. This ensures an efficient, stable introduction of the transposon-based expression vectors into vertebrate host cells in one step, which can then be screened for a desired functional or binding phenotype of the expressed proteins, after which the relevant coding sequences for the expressed proteins, including antibodies and fragments thereof, can be identified by standard cloning and DNA sequencing techniques.

FIELD OF THE INVENTION

Technologies for the identification of specific functional and bindingproteins.

BACKGROUND OF THE INVENTION

The discovery of target-specific proteins, including antibodies andfragments thereof, is of significant commercial interest, because theselection of highly selective functional proteins or binding proteins,including antibodies and fragments thereof, has a high potential for thedevelopment of new biological entities (NBEs) with novel therapeuticproperties that very specifically integrate, or interfere withbiological processes, and therefore are predicted to display lowerside-effect profiles than conventional new chemical entities (NCEs). Inthat respect, particularly the development of highly target-specific,therapeutic antibodies, and antibody-based therapeutics, have paved theway to completely novel therapies with improved efficacy. As aconsequence, therapeutic monoclonal antibodies represent the fastestgrowing segment in the development of new drugs over the last decade,and presently generate about USD 50 billion global revenues, whichaccounts for a significant share of the total global market ofpharmaceutical drugs.

Therefore, efficient and innovative technologies, that allow thediscovery of highly potent, but also well tolerated therapeuticproteins, in particular antibody-based therapeutics, are in high demand.

In order to identify a protein with a desired functionality or aspecific binding property, as is the case for antibodies, it is requiredto generate, to functionally express and to screen large, diversecollections, or libraries of proteins, including antibodies andfragments thereof, for desired functional properties or target bindingspecificity. A number of technologies have been developed over the pasttwenty years, which allow expression of diverse protein libraries eitherin host cells, or on viral and phage particles and methods for theirhigh-throughput screening and/or panning toward a desired functionalproperty, or binding phenotype.

Standard, state-of-the-art technologies to achieve identification oftarget-specific binders or proteins with desired functional propertiesinclude, e.g. phage-display, retroviral display, bacterial display,yeast display and various mammalian cell display technologies, incombination with solid surface binding (panning) and/or other enrichmenttechniques. All of these technologies are covered by various patents andpending patent applications.

While phage and prokaryotic display systems have been established andare widely adopted in the biotech industry and in academia for theidentification of target-specific binders, including antibody fragments(Hoogenboom, Nature Biotechnology 23, 1105-1116 (2005)), they sufferfrom a variety of limitations, including the inability to expressfull-length versions of larger proteins, including full-lengthantibodies, the lack of proper post-translational modification, the lackof proper folding by vertebrate chaperones, and, in the case ofantibodies, an artificially enforced heavy and light chain combination.Therefore, in case of antibody discovery by these methods,“reformatting” into full-length antibodies and mammalian cell expressionis required. Due to the above-mentioned limitations this frequentlyresults in antibodies with unfavorable biophysical properties (e.g. lowstability, tendency to aggregate, diminished affinity), limiting thetherapeutic and diagnostic potential of such proteins. This, on onehand, leads to significant attrition rates in the development of leadmolecules generated by these methods, and, on the other hand, requiressignificant effort to correct the biophysical and molecular liabilitiesin these proteins for further downstream drug development.

Therefore, protein and antibody discovery technologies have beendeveloped using lower eukaryotic (e.g. yeast) and, more recently, alsomammalian cell expression systems for the identification of proteinswith desired properties, as these technologies allow (i) expression oflarger, full-length proteins, including full-length antibodies, (ii)better or normal post-translational modification, and, (iii) in case ofantibodies, proper heavy-light chain pairing (Beerli & Rader, mAbs 2,365-378 (2010)). This, in aggregate, selects for proteins with favorablebiophysical properties that have a higher potential in drug developmentand therapeutic use.

Although expression and screening of proteins in vertebrate cells wouldbe most desirable, because vertebrate cells (e.g. hamster CHO, humanHEK-293, or chicken DT40 cells) are preferred expression systems for theproduction of larger therapeutic proteins, such as antibodies, thesetechnologies are currently also associated with a number of limitations,which has lead to a slow adoption of these technologies in academia andindustry.

First, vertebrate cells are not as efficiently and stably geneticallymodified, as, e.g. prokaryotic or lower eukaryotic cells like yeast.Therefore, its remains a challenge to generate diverse (complex) enoughvertebrate cell based proteins libraries, from which candidates withdesired properties or highest binding affinities can be identified.Second, in order to efficiently isolate proteins with desiredproperties, usually iterative rounds of cell enrichment are required.Vertebrate expression either by transient transfection of plasmids(Higuchi et al. J. Immunol. Methods 202, 193-204 (1997)), or transientviral expression systems, like sindbis or vaccinia virus (Beerli et al.PNAS 105, 14336-14341 (2008), and WO02102885) do not allow multiplerounds of cell selection required to efficiently enrich highly specificproteins, and these methods are therefore either restricted to screeningof small, pre-enriched libraries of proteins, or they do require tediousvirus isolation/cell re-infection cycles.

In order to achieve stable expression of binding proteins and antibodiesin vertebrate cells, that do allow multiple rounds of selections basedon stable genotype-phenotype coupling, technologies have been developed,utilizing specific recombinases (flp/frt recombinase system, Thou et al.mAbs 5, 508-518 (2010)), or retroviral vectors (WO2009109368). However,the flp/frt recombination is a low-efficient system for stableintegration of genes into vertebrate host cell genomes and therefore,again, only applicable to small, pre-selected libraries, or theoptimization of selected protein or antibody candidates.

In comparison to the flp/frt recombinase system, retroviral vectorsallow more efficient stable genetic modification of vertebrate hostcells and the generation of more complex cellular libraries. However,(i) they are restricted to only selected permissible cell lines, (ii)they represent a biosafety risk, when human cells are utilized, (iii)retroviral expression vectors are subject to unwanted mutagenesis of thelibrary sequences due to low-fidelity reverse transcription, (iv)retroviral vectors do not allow integration of genomic expressioncassettes with intact intron/exon structure, due to splicing of theretroviral genome prior to packaging of the vector into retroviralparticles, (v) retroviruses are subject to uncontrollable andunfavorable homologous recombination of library sequences duringpackaging of the viral genomes, (vi) are subject to retroviralsilencing, and (vii) require a tedious two-step packaging-celltransfection/host-cell infection procedure. All these limitationsrepresent significant challenges and limitations, and introducesignificant complexities for the utility of retroviral vector basedapproaches in generating high-quality/high complexity vertebrate celllibraries for efficient target-specific protein, or antibody discovery.

Therefore, clearly a need exists for a more efficient, more controllableand straightforward technology that allows the generation ofhigh-quality and highly complex vertebrate cell based librariesexpressing diverse libraries of proteins including antibodies andfragments thereof from which proteins with highly specific functionand/or binding properties and high affinities can be isolated.

(b) Transposases/Transposition:

Transposons, or transposable elements (TEs), are genetic elements withthe capability to stably integrate into host cell genomes, a processthat is called transposition (Ivics et al. Mobile DNA 1, 25 (2010))(incorporated herein by reference in its entirety). TEs were alreadypostulated in the 1950s by Barbara McClintock in genetic studies withmaize, but the first functional models for transposition have beendescribed for bacterial TEs at the end of the 1970s (Shapiro, PNAS 76,1933-1937 (1979)) (incorporated herein by reference in its entirety).

Meanwhile it is clear that TEs are present in the genome of everyorganism, and genomic sequencing has revealed that approximately 45% ofthe human genome is transposon derived (International Human GenomeSequencing Consortium Nature 409: 860-921 (2001)) (incorporated hereinby reference in its entirety). However, as opposed to invertebrates,where functional (or autonomous) TEs have been identified (FIG. 1a ),humans and most higher vertebrates do not contain functional TEs. It hasbeen hypothesized that evolutionary selective pressure against themutagenic potential of TEs lead to their functional inactivationmillions of years ago during evolution.

Autonomous TEs comprise DNA that encodes a transposase enzyme located inbetween two inverted terminal repeat sequences (ITRs), which arerecognized by the transposase enzyme encoded in between the ITRs andwhich can catalyze the transposition of the TE into any double strandedDNA sequence (FIG. 1a ). There are two different classes of transposons:class I, or retrotransposons, that mobilize via an RNA intermediate anda “copy-and-paste” mechanism (FIG. 2b ), and class II, or DNAtransposons, that mobilize via excision-integration, or a“cut-and-paste” mechanism (FIG. 2a ) (Ivics et al. Nat. Methods 6,415-422 (2009)) (incorporated herein by reference in its entirety).

Bacterial, lower eukaryotic (e.g. yeast) and invertebrate transposonsappear to be largely species specific, and cannot be used for efficienttransposition of DNA in vertebrate cells. Only, after a first activetransposon had been artificially reconstructed by sequence shuffling ofinactive TEs from fish, which was therefore called “Sleeping Beauty”(Ivics et al. Cell 91, 501-510 (1997)) (incorporated herein by referencein its entirety), did it become possible to successfully achieve DNAintegration by transposition into vertebrate cells, including humancells. Sleeping Beauty is a class II DNA transposon belonging to theTc1/mariner family of transposons (Ni et al. Briefings Funct. GenomicsProteomics 7, 444-453 (2008)) (incorporated herein by reference in itsentirety). In the meantime, additional functional transposons have beenidentified or reconstructed from different species, includingDrosophila, frog and even human genomes, that all have been shown toallow DNA transposition into vertebrate and also human host cell genomes(FIG. 3). Each of these transposons, have advantages and disadvantagesthat are related to transposition efficiency, stability of expression,genetic payload capacity, etc.

To date, transposon-mediated technologies for the expression of diverselibraries of proteins, including antibodies and fragments thereof, invertebrate host cells for the isolation of target specific, functionalbinding proteins, including antibodies and fragments thereof, have notbeen disclosed in the prior art.

BRIEF SUMMARY OF THE INVENTION

The method disclosed herein describes a novel technology offeringunparalleled efficiency, flexibility, utility and speed for thediscovery and optimization of polypeptides having a desired bindingspecificity and/or functionality, including antigen-binding moleculessuch as antibodies and fragments thereof, for desired functional and/orbinding phenotypes. The novel method is based on transposable constructsand diverse DNA libraries cloned into transposable vectors and theirtransfection into host cells by concomitant transient expression of afunctional transposase enzyme. This ensures an efficient, stableintroduction of the transposon-based expression vectors into vertebratehost cells in one step, which can then be screened for a desiredfunctional or binding phenotype of the expressed proteins, after whichthe relevant coding sequences for the expressed proteins, includingantibodies and fragments thereof, can be identified by standard cloningand DNA sequencing techniques.

In one embodiment, the invention is broadly directed to a method foridentifying a polypeptide having a desired binding specificity orfunctionality, comprising:

(i) generating a diverse collection of polynucleotides encodingpolypeptides having different binding specificities or functionalities,wherein said polynucleotides comprise a sequence coding for apolypeptide disposed between first and second inverted terminal repeatsequences that are recognized by and functional with a least onetransposase enzyme;(ii) introducing the diverse collection of polynucleotides of (i) intohost cells;(iii) expressing at least one transposase enzyme functional with saidinverted terminal repeat sequences in said host cells so that saiddiverse collection of polynucleotides is integrated into the host cellgenome to provide a host cell population that expresses said diversecollection of polynucleotides encoding polypeptides having differentbinding specificities or functionalities;(iv) screening said host cells to identify a host cell expressing apolypeptide having a desired binding specificity or functionality; and(v) isolating the polynucleotide sequence encoding said polypeptide fromsaid host cell.

In a preferred embodiment, the polynucleotides are DNA molecules. In oneembodiment, the diverse collection of polynucleotides comprises aligand-binding sequence of a receptor, or a target binding sequence of abinding molecule. In a preferred embodiment, the polynucleotidescomprise a sequence encoding an antigen-binding molecule, such as anantibody VH or VL domain, or an antigen-binding fragment thereof, orantibody heavy or light chains that are full-length (i.e., which includethe constant region). In certain embodiments, the polynucleotides maycomprise a sequence encoding both a V_(H) and V_(L) region, or bothantibody heavy and light chains. In another embodiment, thepolynucleotides comprise a sequence encoding a single-chain Fv or a Fabdomain.

In one embodiment, the diverse collection of polynucleotides isgenerated by subjecting V region gene sequences to PCR undermutagenizing conditions, for example, by PCR amplification of V regionrepertoires from vertebrate B cells. In another embodiment, the diversecollection of polynucleotides is generated by gene synthesis (e.g., byrandomization of sequences encoding a polypeptide having known bindingspecificity and/or functionality). In one useful embodiment, the diversecollection of polynucleotides comprises plasmid vectors. In anotheruseful embodiment, the diverse collection of polynucleotides comprisesdouble-stranded DNA PCR amplicons. The plasmid vectors may comprise asequence encoding a marker gene, such as a fluorescent marker, a cellsurface marker, or a selectable marker. The marker gene sequence may beupstream or downstream of the sequence encoding the polypeptide having abinding specificity or functionality, but between the inverted terminalrepeat sequences. Alternatively, the marker gene sequence may bedownstream of said sequence encoding a polypeptide having bindingspecificity or functionality and separated by an internal ribosomalentry site.

In some embodiments, the diverse collection of polynucleotides encode aplurality of antigen-binding molecules of a vertebrate, such as amammal, e.g., a human.

In one embodiment, step (ii) of the method comprises introducing intohost cells polynucleotides comprising sequences encoding immunoglobulinV_(H) or V_(L) regions, or antigen-binding fragments thereof, andwherein said V_(H) and V_(L) region sequences are encoded on separatevectors. In another embodiment, step (ii) of the method of the inventioncomprises introducing into host cells polynucleotides comprisingsequences encoding full-length immunoglobulin heavy or light chains, orantigen-binding fragments thereof, wherein said full-length heavy andlight chain sequences are on separate vectors. The vectors may beintroduced into the host cells simultaneously or sequentially. Inanother embodiment, sequences encoding V_(H) and V_(L) regions orfull-length heavy and light chains are introduced into host cells on thesame vector. In the event that the V_(H) and V_(L) sequences or thefull-length antibody heavy and light chain sequences are introduced intothe host cells on different vectors, it is useful for the invertedterminal repeat sequences on each vector to be recognized by andfunctional with different transposase enzymes.

The host cells are preferably vertebrate cells, and preferably mammaliancells, such as rodent or human cells. Lymphoid cells, e.g., B cells, areparticularly useful. B cells may be progenitor B cells or precursor Bcells such as, for example, Abelson-Murine Leukemia virus transformedprogenitor B cells or precursor B cells and early, immunoglobulin-nullEBV transformed human proB and preB cells. Other useful host cellsinclude B cell lines such as Sp2/0 cells, NSO cells, X63 cells, andAg8653 cells, or common mammalian cell lines such as CHO cells, Per.C6cells, BHK cells, and 293 cells.

In one embodiment of the method of the invention, the expressing step(iii) comprises introducing into said host cells an expression vectorencoding a transposase enzyme that recognizes and is functional with atleast one inverted terminal repeat sequence in the polynucleotides. Thevector encoding the transposase enzyme may be introduced into the hostcells concurrently with or prior or subsequent to the diverse collectionof polynucleotides. In one embodiment, the transposase enzyme istransiently expressed in said host cell. Alternatively, the expressingstep (iii) may comprise inducing an inducible expression system that isstably integrated into the host cell genome, such as, for example, atetracycline-inducible or tamoxifen-inducible system. In a preferredembodiment, step (iii) comprises expressing in the host cell(s) a vectorcomprising a functional Sleeping Beauty transposase or a functionalPiggyBac transposase. In one useful embodiment, step (iii) comprisesexpressing in said host cell a vector comprising SEQ ID NO:11. Inanother useful embodiment, the vector encodes SEQ ID NO:12, or asequence with at least 95% amino acid sequence homology and having thesame or similar inverted terminal repeat sequence specificity.

In another useful embodiment, step (iii) comprises expressing in saidhost cell a vector comprising SEQ ID NO:17. In another usefulembodiment, the vector encodes SEQ ID NO:18, or a sequence with at least95% amino acid sequence homology and having the same or similar invertedterminal repeat sequence specificity.

In one embodiment of the method of the invention, the screening step(iv) comprises magnetic activated cell sorting (MACS), fluorescenceactivated cell sorting (FACS), panning against molecules immobilized ona solid surface panning, selection for binding to cell-membraneassociated molecules incorporated into a cellular, natural orartificially reconstituted lipid bilayer membrane, or high-throughputscreening of individual cell clones in multi-well format for a desiredfunctional or binding phenotype. In one embodiment, the screening step(iv) comprises screening to identify polypeptides having a desiredtarget-binding specificity or functionality. In a preferred embodiment,the screening step (iv) comprises screening to identify antigen-bindingmolecules having desired antigen specificity. In one useful embodiment,the screening step further comprises screening to identifyantigen-binding molecules having one or more desired functionalproperties. The screening step (iv) may comprise multiple cellenrichment cycles with host cell expansion between individual cellenrichment cycles.

In one embodiment of the method of the invention, the step (v) ofisolating the polynucleotide sequence encoding the polypeptide having adesired binding specificity or functionality comprises genomic or RT-PCRamplification or next-generation deep sequencing. In one usefulembodiment, the polynucleotide sequence isolated in step (v) issubjected to affinity optimization. This can be done by subjecting theisolated polynucleotide sequence to PCR or RT-PCR under mutagenizingconditions. In another useful embodiment, the mutagenized sequence isthen further subjected to steps (i)-(v) of the method of the invention.In a preferred embodiment, the polynucleotide sequence obtained in (v)comprises a sequence encoding a V_(H) or V_(L) region of an antibody, oran antigen-binding fragment thereof, and wherein said antibodyoptimization comprises introducing one or more mutations into acomplementarity determining region or framework region of said V_(H) orV_(L).

In one useful embodiment, the inverted terminal repeat sequences arefrom the PiggyBac transposon system and are recognized by and functionalwith the PiggyBac transposase. In one embodiment, the sequence encodingthe upstream PiggyBac inverted terminal repeat sequence comprises SEQ IDNO:1. In another embodiment, the sequence encoding the downstreamPiggyBac inverted terminal repeat sequence comprises SEQ ID NO:2.

In another useful embodiment, the inverted terminal repeat sequences arefrom the Sleeping Beauty transposon system and are recognized by andfunctional with the Sleeping Beauty transposase. In one embodiment, thesequence encoding the upstream Sleeping Beauty inverted terminal repeatsequence comprises SEQ ID NO:14. In another embodiment, the sequenceencoding the downstream Sleeping Beauty inverted terminal repeatsequence comprises SEQ ID NO:15.

In one embodiment of the invention, the polynucleotides comprise V_(H)or V_(L) region sequences encoding a sequence derived from a humananti-TNF alpha antibody. In one embodiment, the human anti-TNF alphaantibody is D2E7.

In a useful embodiment, step (iii) comprises introducing into said hostcell a vector comprising a sequence encoding a functional PiggyBactransposase. In one embodiment the vector comprises SEQ ID NO:11. Inanother embodiment, the vector encodes SEQ ID NO:12, or a sequence withat least 95% amino acid sequence homology and having the same or similarinverted terminal repeat sequence specificity.

In another useful embodiment, step (iii) comprises expressing in saidhost cell a vector comprising SEQ ID NO:17. In another usefulembodiment, the vector encodes SEQ ID NO:18, or a sequence with at least95% amino acid sequence homology and having the same or similar invertedterminal repeat sequence specificity.

In preferred embodiments, the inverted terminal repeat sequences arerecognized by and functional with at least one transposase selected fromthe group consisting of: PiggyBac, Sleeping Beauty, Frog Prince, Himar1,Passport, Minos, hAT, Tol1, Tol2, Ac/Ds, PIF, Harbinger, Harbinger3-DR,and Hsmar1.

The present invention is further directed to a library of polynucleotidemolecules encoding polypeptides having different binding specificitiesor functionalities, comprising a plurality of polynucleotide molecules,wherein said polynucleotide molecules comprise a sequence encoding apolypeptide having a binding specificity or functionality disposedbetween inverted terminal repeat sequences that are recognized by andfunctional with at least one transposase enzyme. Preferably thepolynucleotides are DNA molecules and comprise a ligand-binding sequenceof a receptor or a target-binding sequence of a binding molecule. In aparticularly preferred embodiment, the library comprisespolynucleotides, wherein each polynucleotide comprises a sequenceencoding an antigen-binding sequence of an antibody. In one embodiment,the library comprises polynucleotides encoding a V_(H) or V_(L) regionof an antibody or an antigen-binding fragment thereof. Alternatively,the polynucleotides may encode a V_(H) region and a V_(L) region. In apreferred embodiment, the polynucleotides of the library comprise asequence encoding a full-length antibody heavy or light chain (i.e.,including the constant region) or an antigen-binding fragment thereof.Alternatively, the polynucleotides may encode both a full-lengthimmunoglobulin heavy and light chain. In other embodiments, thepolynucleotides of the library comprise a sequence encoding asingle-chain Fv or a Fab domain. In preferred embodiments, thepolynucleotides of the library are in the form of plasmids or doublestranded DNA PCR amplicons. In certain embodiments, the plasmids of thelibrary comprise a marker gene. In another embodiment, the plasmidscomprise a sequence encoding a transposase enzyme that recognizes and isfunctional with the inverted terminal repeat sequences. In oneembodiment, the library of the invention comprises polynucleotides thatencode the full-length immunoglobulin heavy chain including the naturalintron/exon structure of an antibody heavy chain. The full-lengthimmunoglobulin heavy chain may comprise the endogenous membrane anchordomain.

The present invention is also directed to a method for generating alibrary of transposable polynucleotides encoding polypeptides havingdifferent binding specificities or functionality, comprising (i)generating a diverse collection of polynucleotides comprising sequencesencoding polypeptides having different binding specificities orfunctionalities, wherein said polynucleotides comprise a sequenceencoding polypeptide having a binding specificity or functionalitydisposed between inverted terminal repeat sequences that are recognizedby and functional with a least one transposase enzyme.

The present invention is also directed to a vector comprising a sequenceencoding a V_(H) or V_(L) region of an antibody, or antigen-bindingportion thereof, disposed between inverted terminal repeat sequencesthat are recognized by and functional with at least one transposaseenzyme. In certain embodiments, the vector encodes a full-length heavyor light chain of an immunoglobulin. Preferably, the sequence encodingthe V_(H) or V_(L) or the heavy or light chain is a randomized sequencegenerated by, for example, PCR amplification under mutagenizingconditions or gene synthesis. In one embodiment, the vector comprisesinverted terminal repeat sequences that are recognized by and functionalwith the PiggyBac transposase. In another embodiment, the invertedterminal repeat sequences are recognized by and functional with theSleeping Beauty transposase. In one embodiment, the vector comprises aVH or VL region sequence derived from an anti-TNF alpha antibody suchas, for example, D2E7. In certain embodiments, the vector comprises atleast one sequence selected from the group consisting of: SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:16, SEQ ID NO:17, and SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

The present invention is also directed to a host cell comprising avector of the invention as described above. In a preferred embodiment,the host cell further comprises an expression vector comprising asequence encoding a transposase that recognizes and is functional withat least one inverted terminal repeat sequence in the vector encodingsaid V_(H) or V_(L) region sequence.

The present invention is still further directed to antigen-bindingmolecules, e.g., antibodies, produced by a method comprising claim 1.

The present invention is also directed to a method for generating apopulation of host cells capable of expressing polypeptides havingdifferent binding specificities or functionalities, comprising:

-   -   (i) generating a diverse collection of polynucleotides        comprising sequences encoding polypeptides having different        binding specificities or functionalities, wherein said        polynucleotides comprise a sequence encoding a polypeptide        having a binding specificity or functionality disposed between        inverted terminal repeat sequences that are recognized by and        functional with a least one transposase enzyme; and    -   (ii) introducing said diverse collection of polynucleotides into        host cells.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: a.) This drawing depicts the configuration of an autonomoustransposable element (TE), which can transpose or “jump” into any targetDNA sequence. The key components of a TE are an active transposaseenzyme that recognizes the inverted terminal repeats (ITRs) flanking thetransposase enzyme itself up- and downstream of its sequence. TEscatalyze either the copying or the excision of the TE, and theintegration in unrelated target DNA sequences. b.) This drawing depictsthe configuration of a transposon vector system, in which the expressionof an active transposase enzyme is effected by an expression vector thatis not coupled to the TE itself. Instead, the TE may contain anysequence(s), or gene(s) of interest that is/are cloned in between theup- and downstream ITRs. Integration of the TE containing anysequence(s), or gene(s) of interest (e.g. a DNA library encoding alibrary of proteins) may integrate into unrelated target DNA sequences,if the transposase enzyme expression is provided in trans, e.g. by aseparate transposase expression construct, as depicted here.

FIG. 2: a) This drawing depicts the two different ways how TEs can“jump” or transpose into unrelated target DNA. For group II transposons,the transposase enzyme in a first step recognizes the ITRs of thetransposable element and catalyzes the excision of the TE from DNA. In asecond step, the excised TE is inserted into unrelated target DNAsequence, which is also catalyzed by the transposase enzyme. Thisresults in a “cut-and-paste” mechanism of transposition. For group Itransposons (shown in b.) the coding information of the TE is firstreplicated (e.g. transcribed and reverse transcribed, in the case ofretrotransposons) and the replicated TE then integrates into unrelatedtarget DNA sequence, which is catalyzed by the transposase enzyme. Thisresults in a “copy-and-paste” mechanism of transposition.

FIG. 3: This figure provides an overview of active transposase enzymesthat have been identified and/or reconstructed from dormant, inactiveTEs, and that have been shown to be able to confer transposition invarious vertebrate and also human cells, as provided in the table. Thetable has been adapted from Table I of publication Ni et al. BriefingsFunctional Genomics Proteomics 7, 444-453 (2008) (incorporated herein byreference in its entirety)

FIG. 4: This figure outlines the principle of the method disclosedherein, for the isolation of coding information for proteins, includingantibodies and fragments thereof, with a desired function, e.g. thebinding to a target of interest, as depicted here. The gene(s) ofinterest, e.g. a diverse transposable DNA library encoding proteins,including antibody polypeptide chains or fragments thereof, that iscloned in between inverted terminal repeats (ITRs) of a transposableconstruct is introduced into a vertebrate host cell together with anexpression vector for an active transposase enzyme (see top of thedrawing). The expression of the transposon enzyme in said host cells intrans and the presence of the gene(s) of interest cloned in between ITRsthat can be recognized by the transposase enzyme allows the stableintegration of the ITR-flanked gene(s) of interest into the genome ofthe host cells, which can then stably express the protein(s) of interestencoded by the genes of interest. The cellular library expressing theprotein(s) of interest can then be screened for a desired functionalityof the expressed proteins, e.g., but not limited to the binding to atarget protein of interest, as depicted here. By means of cellseparation techniques known in the art, e.g. MACS or FACS, the cellsexpressing the protein(s) of interest with the desired phenotype andwhich therefore contain the corresponding genotype, can be isolated andthe coding information for the gene(s) of interest can be retrieve fromthe isolated cells by cloning techniques known in the art, e.g. but notlimited to genomic PCR cloning, as depicted here.

FIGS. 5a ) and 5 b): This drawing outlines the cloning strategy for thegeneration of a transposable human immunoglobulin (Ig) kappa light chain(LC) expression vector, as described in Example 1. FIG. 5 a.) depictsthe cloning strategy for the insertion of 5′- and 3′-ITRs from thePiggyBac transposon into the mammalian expression vector pIRES-EGFP(Invitrogen, Carlsbad, Calif., USA), which already contains the strongmammalian cell promoter element pCMV(IE) (immediate early promoter ofCMV), and intron/polyA signals for strong mammalian host cellexpression. In addition, downstream of the ClaI, EcoRV, NotI, EcoRIcontaining multiple cloning site, into which gene(s) of interest can becloned, pIRES-EGFP contains an internal ribosomal entry site (IRES) witha downstream ORF of enhanced green fluorescent protein (EGFP), whicheffects the coupling of expression of gene(s) of interest clonedupstream of the IRES. Bacterial functional elements (ampicillinresistance gene, amp^(R)) and a bacterial origin of replication (Col E1)for amplification and selection of the plasmid in E. coli are depictedas well. The resulting PiggyBac ITRs containing plasmid is designatedpIRES-EGFP-T1T2. FIG. 5b ) then depicts the insertion of a genesynthesized human Ig kappa LC into the unique EcoRV restriction enzymesite of pIRES-EGFP-T1-T2, which positions the human Ig kappa LC upstreamof the IRES-EGFP cassette, and thereby couples the expression of thehuman Ig kappa LC to EGFP marker gene expression. The insertion of thehuman Ig kappa LC results in transposable human Ig kappa LC expressionvector pIRES-EGFP-T1T2-IgL. The drawings show selected uniquerestriction enzyme sites in the plasmids, as well as selected duplicatedsites resulting from cloning steps.

FIG. 6: This drawing outlines the cloning of a transposable humanimmunoglobulin (Ig) gamma 1 heavy chain (HC) expression vector, whichcan be generated by exchange of the human Ig kappa LC open reading frame(ORF) against the ORF for a human Ig gamma 1 HC ORF. The design of thefinal Ig gamma 1 HC ORF is similar, also with regard to the engineeringof a unique Eco47III restriction enzyme site separating the variable (V)from the constant (C) coding regions, which allows the exchange of asingle antibody V coding region against a diverse library of antibody Vcoding regions, as described in Example 3.

FIG. 7: This drawing depicts the cloning of a mammalian PiggyBactransposase enzyme expression vector, as described in the Example 4,using pCDNA3.1(+) hygro as the backbone of the mammalian expressionvector, into which the gene synthesized ORF from PiggyBac transposase iscloned into the unique EcoRV restriction enzyme site of pCDNA3.1(+)hygro, resulting in PiggyBac transposon expression vector pCDNA3.1(+)hygro-PB expression vector pCDNA3.1(+) hygro-PB. Also in this drawingthe relative position of other mammalian functional elements (CMV-IEpromoter, BGH-polyA signal, SV40-polyA segment, hygromycin B ORF) andbacterial functional elements (ampicillin resistance gene, ampR, originof replication, ColE1), as well as selected relevant restriction enzymerecognition sites are shown.

FIG. 8: This drawing depicts the cloning of a Sleeping Beautytransposable human immunoglobulin kappa light chain (Ig-kappa LC)expression vector, as described in Example 5. The cloning can beperformed by sequentially replacing the PiggyBac 5′ and 3′ ITRs withSleeping Beauty 5′ and 3′ITRs in construct pIRES-EGFP-T1T2-IgL. Also inthis drawing the relative position of other mammalian functionalelements (CMV-IE promoter, BGH-polyA signal, SV40-polyA segment,hygromycin B ORF) and bacterial functional elements (ampicillinresistance gene, ampR, origin of replication, ColE1), as well asselected relevant restriction enzyme recognition sites are shown.

FIG. 9: This drawing depicts the cloning of a mammalian Sleeping Beautytransposase enzyme expression vector, as described in the Example 6,using pCDNA3.1(+) hygro as the backbone of the mammalian expressionvector, into which the gene synthesized ORF from Sleeping Beautytransposase is cloned into the unique EcoRV restriction enzyme site ofpCDNA3.1(+) hygro, resulting in Sleeping Beauty transposon expressionvector pCDNA3.1(+) hygro-SB. Also in this drawing the relative positionof other mammalian functional elements (CMV-IE promoter, BGH-polyAsignal, SV40-polyA segment, hygromycin B ORF) and bacterial functionalelements (ampicillin resistance gene, ampR, origin of replication,ColE1), as well as selected relevant restriction enzyme recognitionsites are shown.

FIG. 10: This drawing shows the arrangement of functional elements andposition of selected unique restriction enzyme sites within thegene-synthesized DNA fragments 1.) and 2.) that were utilized in Example4, in order to clone both “empty” IgH chain expression vectors allowingtransposition utilizing either PiggyBac or Sleeping Beauty transposase.The origin of the functional elements is disclosed in detail in thedescription of the Example.

FIG. 11: This drawing shows the final design and plasmid map of thetransposable expression vectors for human, membrane bound Ig-gamma1heavy chains (left) and human Ig kappa light chains (right). For the IgHexpression vector, the V_(H)-coding region may be replaced by V_(H)coding regions of any other monoclonal antibody, or by a V_(H)-genelibrary, using unique restriction enzyme sites NotI and NheI, flankingthe V_(H) coding region in this vector. For the IgL expression vector,the V_(L)-coding region may be replaced by V_(L) coding regions of anyother monoclonal antibody, or by a V_(L)-gene library, using uniquerestriction enzyme sites NotI and BsiWI, flanking the V_(L) codingregion in this vector. The 8 vector constructions for PiggyBac andSleeping Beauty transposable IgH and IgL vectors, disclosed in detail inExample 4 all share this general design. The two vector maps displayedhere correspond to the vector maps of pPB-EGFP-HC-Ac10 (left) andpPB-EGFP-LC-Ac10 (right), and the additional vectors for hBU12 heavychain (HC) or light chain (LC), containing either PiggyBac or SleepingBeauty ITRs, are provided in the tables below. Sequences of all vectorsin this figure are provided in Example 4.

FIG. 12: This figure shows two dimensional FACS dot-plots, in which thesurface expression of human IgG from transfected and transposed IgHC andIgLC expression vectors is detected on the surface of 63-12 A-MuLVtransformed murine proB cells derived from RAG-2-deficient mice. d2 postTF means that the FACS analysis was performed 2 days after transfectionof vector constructs into 63-12 cells. The FACS plots in the left-handcolumn represents negative and positive controls for the transfection.NC=mock electroporation of cells without plasmid DNA.pEGFP-N3=transfection control with pEGFP-N3 control vector, whichcontrols for the transfection efficiency by rendering transfected cellsgreen. The second column from the left shows FACS plots from 63-12 cellsco-transfected with either PiggyBac-transposase vector,pPB-EGFP-HC-Ac10, pPB-EGFP-LC-Ac10 vectors (top row), orPiggyBac-transposase vector, pPB-EGFP-HC-hBU12, pPB-EGFP-LC-hBU12vectors (middle row), or with Sleeping Beauty-transposase vector,pSB-EGFP-HC-Ac10, pSB-EGFP-LC-Ac10 vectors (bottom row). The second-leftcolumn labeled “d2 post TF” shows the analysis for cells expressing IgGon the cell surface (Y-axis) and EGFP expression (X-axis) two days postco-transfection of the vectors as mentioned above. Surface IgG and EGFPdouble positive cells were FACS sorted as indicated by the rectangulargate. The second-right column labeled “d9 1× sorted” shows the analysisof surface IgG and EGFP expression in the cell population that wassorted at day 2 after transfection, analyzed in the same way. Sortinggates for the second FACS sort are also provided as rectangular gates.The rightmost column labeled “d16 2× sorted” shows the analysis ofsurface IgG and EGFP expression of the cell populations that had beenre-sorted at day 9 after transfection, and analyzed in the same way forsurface IgG and EGFP expression as in the previous experiments.

FIG. 13: This figure depicts the demonstration that proB cellsexpressing CD30-specific IgG on the surface of 63-12 cells canspecifically be stained and detected by CD30 antigen, and that theCD30-specific cells be detected and re-isolated from a large populationof cells expressing surface IgG of unrelated specificity (here CD19), inwhich the CD30-specific cells have been spiked in with decreasingfrequency. The FACS dot-plot on top shows the detection of IgG (viaanti-kappaLC staining) and CD30 binding (via CD30-antigen staining) onthe surface of the positive control cells, which are 63-12 cells stablytransposed and 2× sorted for expressing human anti-CD30 IgG, clone Ac10on the cell surface. As expected, a quite pure population (97.3%) ofIgG-positive/CD30-reactive was detectable in the upper right quadrant ofthe FACs-dot-plot. The numbers on top of each FACS-plot indicates thenumber of live cells based on FSC/SSC gating that were acquired in eachexperiment. The middle row shows the FACS analysis for IgG-positive/CD30reactive cells detectable in a background of IgGpositive/CD19 specificcells. The number above the number of events indicates the dilutionfactor of anti-CD30 specific IgG positive cells that were used for thegeneration of the “spiked-in” population of anti-CD30 mAb IgG positivecells in a background of anti-CD19 mAb IgG positive cells. The sortinggates are indicated that were used to specifically isolateIgG-positive/CD30 antigen reactive cells from the spiked-in populations.Larger numbers of events needed to be acquired in order to allowdetection and isolation of the IgG-positive/CD30 cells at higherdilutions. The lower row of FACS plots then shows the re-analysis ofsorted cells after the cells had been expanded for 12 days for the sameparameters (IgG-expression & CD30 antigen specificity).

FIG. 14: This figure shows the cloning of a transposable vector for ahuman Ig-gamma1 heavy chain (HC) in genomic configuration. The linearfragment on top represents the human gamma1 exon and introns formembrane-bound Ig-gamma1-HC, with flanking NheI and BstBI restrictionsites added to allow ligation into Ig-gamma1 HC cDNA vectorpPB-EGFP-HC-Ac10. H-designates the Hinge-region exon, M1 and M2represent the exons encoding the trans-membrane region of surfaceexpressed Ig heavy chain. With a simple one-step ligation the cDNAC-gamma1 region of the transposable human heavy chain vector is replacedby its genomic counterpart as indicated in the figure. Using thisstrategy, the V_(H) coding region will be ligated in-frame to the C_(H)1coding exon of human C-gamma1.

FIG. 15: This figure shows the sequence and overall design of the kappalight chain library. CDR3 coding region is underlined. Usefulrestriction sites are indicated.

FIG. 16: This figure shows the sequence and overall design of the gammaheavy chain library, showing as an example the library fragmentrandomized using the NNK4 randomization strategy. The gamma heavy chainlibrary fragments randomized using the NNK6, NNK8 and NNK10randomization strategies differ only in the number of randomized aminoacid residues in the HCDR3 region. HCDR3 coding region is underlined.The ARG codon encodes Lysine and Arginine. Useful restriction sites areindicated.

FIG. 17: This figure shows the digestion of PCR templates prior toamplification with primers. (A) Digestion of pUC57_Jkappa2-Ckappa withthe restriction endonuclease ScaI produces a blunt-ended DNA fragmentideal for priming with the primer LCDR3-NNK6-F. (B) Digestion ofpUC57_J_(H)4 with the restriction endonuclease DrdI produces a DNAfragment ideal for priming with the primers HCDR3-NNK4-F, HCDR3-NNK6-F,HCDR3-NNK8-F, and HCDR3-NNK10-F

FIG. 18: This figure shows the electropherograms spanning the randomizedLCDR3 and HCDR3 region of the PCR amplicons generated to diversify theLCDR3 region by the NNK-6 approach for Vkappa (A), and the HCDR3 regionby the NNK4-approach for V_(H), as disclosed in Examples 12 and 13,respectively.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “diverse collection” means a plurality of variants ormutants of particular functional or binding proteins exhibitingdifferences in the encoding nucleotide sequences or in the primary aminoacid sequences, which define different functionalities or bindingproperties.

As used herein, “library” means a plurality of polynucleotides encodingpolypeptides having different binding specificities and/orfunctionalities. In certain embodiments, the library may comprisepolynucleotides encoding at least 10², at least 10³, at least 10⁴, atleast 10⁵, at least 10⁶, at least 10⁷, at least 10⁸, or at least 10⁹unique polypeptides, such as, for example, full-length antibody heavy orlight chains or VH or VL domains.

As used herein, “inverted terminal repeat sequence” or “ITR” means asequence identified at the 5′ or 3′ termini of transposable elementsthat are recognized by transposases and which mediate the transpositionof the ITRs including intervening coding information from one DNAconstruct or locus to another DNA construct or locus.

As used herein, “transposase” means an enzyme that has the capacity torecognize and to bind to ITRs and to mediate the mobilization of atransposable element from one target DNA sequence to another target DNAsequence.

As used herein, “antigen binding molecule” refers in its broadest senseto a molecule that specifically binds an antigenic determinant. Anon-limiting example of an antigen binding molecule is an antibody orfragment thereof that retains antigen-specific binding. By “specificallybinds” is meant that the binding is selective for the antigen and can bediscriminated from unwanted or nonspecific interactions.

As used herein, the term “antibody” is intended to include wholeantibody molecules, including monoclonal, polyclonal and multispecific(e.g., bispecific) antibodies, as well as antibody fragments having anFc region and retaining binding specificity, and fusion proteins thatinclude a region equivalent to the Fc region of an immunoglobulin andthat retain binding specificity. Also encompassed are antibody fragmentsthat retain binding specificity including, but not limited to, VHfragments, VL fragments, Fab fragments, F(ab′)₂ fragments, scFvfragments, Fv fragments, minibodies, diabodies, triabodies, andtetrabodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134(2003)) (incorporated by reference in its entirety).

An embodiment of the invention disclosed herein is a method for theidentification of specific functional or binding polypeptides,including, but not limited to antibody chains or fragments thereof (FIG.4), which comprises:

-   i. cloning of diverse transposable DNA libraries encoding proteins,    including antibody polypeptide chains or fragments thereof, in    between inverted terminal repeats (ITRs) derived from transposable    elements and recognizable by and functional with at least one    transposase enzyme,-   ii. introduction of one or more diverse transposable DNA libraries    of step (i) into vertebrate host cells by standard methods known in    the art,-   iii. providing temporary expression of at least one functional    transposase enzyme in said vertebrate host cells in trans, such that    said one or more diverse transposable DNA libraries are stably    integrated into the vertebrate host cell genomes, thereby providing    a vertebrate host cell population that then stably expresses diverse    libraries of proteins, including antibody chains or fragments    thereof,-   iv. screening of said diverse cellular libraries, stably expressing    proteins, including antibodies or fragments thereof, for a desired    functional or binding phenotype by methods known in the art,-   v. optionally, including iterative enrichment cycles with the stably    genetically modified vertebrate host cells for a desired binding or    functional phenotype, and-   vi. isolation of the corresponding genes from the enriched host    cells encoding the desired binding or functional phenotype by    standard cloning methods, known in the art, for instance, but not    limited to, PCR (polymerase chain reaction), using primers specific    for the sequences contained in the one or more transposed DNA    library constructs.

A preferred embodiment of step (i) is to generate diverse transposableDNA libraries either by gene synthesis, or by polymerase chain reaction(PCR) using appropriate primers for the amplification of diverse proteincoding regions, and DNA templates comprising a diversity of bindingproteins, including antibodies, or fragments thereof, by methods knownin the art.

For the generation of diverse antibody libraries, a diverse collectionof antibody heavy and light chain sequences may be generated by standardgene synthesis in which the V region coding sequences may be randomizedat certain positions, e.g. but not limited to, any or all of thecomplementarity determining regions (CDRs) of the antibody heavy orlight chain V-regions. The diversity can be restricted to individualCDRs of the V-regions, or to a particular or several frameworkpositions, and/or to particular positions in one or more of the CDRregions. The V regions with designed variations, as described above, canbe synthesized as a fragment encoding entire antibody heavy or lightchains that are flanked by inverted terminal repeats functional for atleast one desired transposase enzyme. Preferably, the DNA librarycontaining diverse variable domains encoding V regions for antibodyheavy or light chains is generated, and flanked by appropriate cloningsites, including but not limited to restriction enzyme recognitionsites, that are compatible with cloning sites in antibody heavy or lightchain expression vectors. Useful transposon expression systems for usein the methods of the invention include, for example, the PiggyBactransposon system as described, for example, in U.S. Pat. Nos.6,218,185; 6,551,825; 6,962,810; 7,105,343; and 7,932,088 (the entirecontents of each of which are hereby incorporated by reference) and theSleeping Beauty transposon system as described in U.S. Pat. Nos.6,489,458; 7,148,203; 7,160,682; US 2011 117072; US 2004 077572; and US2006 252140 (the entire contents of each of which are herebyincorporated by reference.)

Diverse antibody heavy and light chain libraries may also be obtainedfrom B cell populations isolated from desired vertebrate species,preferably humans, and preferably from cellular compartments containingB cells, e.g., but not limited to peripheral or cord blood, and lymphoidorgans like bone marrow, spleen, tonsils and lymph-node tissues. In thiscase, diverse antibody V region sequences for antibody heavy and lightchains can be isolated by RT-PCR or by genomic PCR using antibody heavyand light chain specific degenerate PCR primer pairs, that can amplifythe majority of V-region families by providing upstream primers thatbind to homologous sequences upstream of, or within leader sequences,upstream of or within V-region frameworks, and by providing downstreamprimers that bind in regions of homology within or downstream of the Jjoining gene segment of variable domain coding regions, or within ordownstream of the coding regions of the constant regions of antibodyheavy or light chains.

The PCR primer sets utilized for the amplification of diverse variablecoding regions may be flanked by appropriate cloning sites, e.g. but notlimited to restriction enzyme recognition sites, that are compatiblewith cloning sites in antibody heavy or light chain expression vectors.

The transposable DNA libraries of step (i) encoding diverse proteins,including antibodies and antibody fragments thereof, can be provided inthe form of plasmid libraries, in which the gene-synthesized or the PCRamplified transposable DNA libraries are cloned using appropriatecloning sites, as mentioned above. Alternatively, the transposable DNAlibraries encoding diverse libraries of binding proteins, such asantibodies and fragments thereof, can be provided in form of linear,double-stranded DNA constructs, directly as a result of DNA synthesis,or as a result of PCR amplification. The latter approach of providingthe transposable DNA libraries as linear double-stranded DNA PCRamplicons, that have not been cloned into expression vectors or plasmids(in comparison to all other vertebrate cell expression systems) has theadvantage that the maximum molecular complexity of the transposable DNAlibraries is maintained and not compromised by a limited cloning orligation efficiency into an expression vector. In contrast, cloning byligation, or otherwise, into plasmid expression or shuttle vectors is anecessary intermediate for all other plasmid-based or viral vector basedvertebrate cell expression systems.

However, the use of plasmid-based transposon expression vectorscontaining the diverse transposable DNA libraries encoding diversebinding proteins, including antibodies and antibody fragments thereof,has the advantage that these expression vectors can be engineered tocontain additional functional elements, that allow the screening, or,alternatively, the selection for stably transposed vertebrate host cellsfor the stable integration of the transposon expression vector intransposed vertebrate host cells.

This is achieved by providing in operable linkage to the diversetransposable DNA libraries, i.e. cloned into the transposon expressionvectors in cis, expression cassettes for marker genes including, but notlimited to, fluorescent marker proteins (e.g. green, yellow, red, orblue fluorescent proteins, and enhanced versions thereof, as known inthe art), or expression cassettes for cell surface markers including,but not limited to, CD markers, against which specific diagnosticantibodies or other diagnostic tools are available.

Alternatively, expression cassettes for selectable markers, that allowselection of transposed vertebrate host cells for antibiotic resistance,including, but not limited to, puromycin, hygromycinB, bleomycin,neomycin resistance, can be provided in operable linkage to the diversetransposable DNA libraries, i.e. cloned into the transposon expressionvectors in cis.

The operable linkage can be achieved by cloning of said expressioncassettes for marker genes or antibiotic resistance markers, either up-or downstream of the coding regions comprising said diverse transposableDNA libraries, but within the inverted terminal repeats of thetransposon vector.

Alternatively, the operable linkage can be achieved by cloning of thecoding regions for said marker or antibiotic resistance genes downstreamof the coding regions comprising said diverse transposable DNAlibraries, but separated by internal ribosomal entry site (IRES)sequences, that ensure transcriptional coupling of the expression ofsaid diverse transposable DNA libraries with said marker or antibioticresistance genes, and thereby allowing the screening for or selection ofstably transposed vertebrate host cells.

In step (ii) of the method disclosed herein, said diverse transposableDNA libraries encoding diverse libraries of proteins, includingantibodies and fragments thereof, are introduced into desired vertebratehost cells by methods known in the art to efficiently transfer DNAacross vertebrate cell membranes, including, but not limited to,DNA-transfection using liposomes, Calcium phosphate, DEAE-dextran,polyethyleneimide (PEI) magnetic particles, or by protoplast fusion,mechanical transfection, including physical, or ballistic methods (genegun), or by nucleofection. Any of the above-mentioned methods and otherappropriate methods to transfer DNA into vertebrate host cells may beused individually, or in combination for step (ii) of the methoddisclosed herein.

In the case of dimeric proteins, including, but not limited to,antibodies and fragments thereof, it is a useful embodiment of themethod disclosed herein to introduce diverse transposable DNA librariesand/or transposon vectors for antibody heavy or light chains containedin separate transposable vectors, which can independently be introducedinto the vertebrate host cells. This either allows the sequentialintroduction of diverse transposable DNA libraries for antibody heavy orlight chains into said cells, or their simultaneous introduction ofdiverse transposable DNA libraries for antibody heavy or light chains,which, in either case, allows the random shuffling of any antibody heavywith any antibody light chain encoded by the at least two separatediverse transposable DNA libraries.

Another useful embodiment of the previous embodiment is to utilizeseparate transposon vectors and/or diverse DNA transposable librariesfor antibody heavy and light chains, where said constructs or librariesare contained on transposable vectors recognized by differenttransposase enzymes (FIG. 3). This allows the independent transpositionof antibody heavy and antibody light chain constructs withoutinterference between the two different transposase enzymes, as onetransposable vector is only recognized and transposed by its specifictransposase enzyme. In case of sequential transposition of transposablevectors or DNA libraries encoding antibody heavy or light chains, theadvantage of utilizing different transposase enzymes with different ITRsequences is, that upon the second transposition event, the firstalready stably transposed construct is not again mobilized for furthertransposition.

This embodiment also allows the discovery of antibodies by the method ofguided selection (Guo-Qiang et al. Methods Mol. Biol. 562, 133-142(2009)) (incorporated herein by reference in its entirety). Guidedselection can e.g. be used for the conversion of any non-human antibodyspecific for a desired target/epitope specificity and with a desiredfunctionality into a fully human antibody, where the same target/epitopespecificity and functionality is preserved. The principle of guidedselection entails the expression of a single antibody chain (heavy orlight chain) of a reference (the “guiding”) antibody, in combinationwith a diverse library of the complementary antibody chains (i.e. light,or heavy chain, respectively), and screening of these heavy-light chaincombinations for the desired functional or binding phenotype. This way,the first antibody chain, “guides” the selection of one or morecomplementary antibody chains from the diverse library for the desiredfunctional or binding phenotype. Once the one or more novelcomplementary antibody chains are isolated, they can be cloned inexpression vectors and again be used to “guide” the selection of thesecond, complementary antibody chain from a diverse antibody chainlibrary. The end-result of this two-step process is that both originalantibody heavy and light chains of a reference antibody are replaced byunrelated and novel antibody chain sequences from the diverse libraries,but where the novel antibody heavy-light chain combination exhibits thesame, or similar functional or binding properties of the originalreference antibody. Therefore, this method requires the ability toindependently express antibody heavy and light chain constructs orlibraries in the vertebrate host cells, which can be achieved by thepreferred embodiment to provide antibody heavy and light chainexpression cassettes in different transposable vector systems,recognized by different transposon enzymes.

However, diverse transposable DNA libraries can also be constructed in away, that the coding regions for multimeric proteins, includingantibodies and fragments thereof, are contained in the same transposonvector, i.e. where the expression of the at least two different subunitsof a multimeric protein, for example V_(H) and V_(L) regions orfull-length heavy and light chains, is operably linked by cloning of therespective expression cassettes or coding regions into the sametransposable vector.

Useful vertebrate host cells for the introduction of transposableconstructs and/or transposable DNA libraries of step (ii) are cells fromvertebrate species that can be or that are immortalized and that can becultured in appropriate cell culture media and under conditions known inthe art. These include, but are not limited to, cells from e.g. frogs,fish, avians, but preferably from mammalian species, including, but notlimited to, cells from rodents, ruminants, non-human primate species andhumans, with cells from rodent or human origin being preferred.

Useful cell types from the above-mentioned species include, but are notlimited to cells of the lymphoid lineage, which can be cultured insuspension and at high densities, with B-lineage derived cells beingpreferred, as they endogenously express all the required proteins,factors, chaperones, and post-translational enzymes for optimalexpression of many proteins, in particular of antibodies, orantibody-based proteins. Of B-lineage derived vertebrate cells, thoseare preferred that represent early differentiation stages, and are knownas progenitor (pro) or precursor (pre) B cells, because said pro- orpreB cells in most cases do not express endogenous antibody chains thatcould interfere with exogenous or heterologous antibody chain expressionthat are part of the method disclosed herein.

Useful pro- and pre-B lineage cells from rodent origin areAbelson-Murine Leukemia virus (A-MuLV) transformed proB and preB cells(Alt et al. Cell 27, 381-390 (1981) (incorporated herein by reference inits entirety)) that express all necessary components for antibodyexpression and also for their proper surface deposition, including the Bcell receptor components Ig-alpha (CD79a, or mb-1), and Ig-beta (CD79b,or B-29) (Hombach et al. Nature 343, 760-762 (1990)) (incorporatedherein by reference in its entirety), but as mentioned above, mostlylack the expression of endogenous antibody or immunoglobulin chains.Here, A-MuLV transformed pro- and preB cells are preferred that arederived from mouse mutants, including, but not limited to, mouse mutantsdefective in recombination activating gene-1 (RAG-1), or recombinationactivating gene-2 (RAG-2), or animals carrying other mutations in genesrequired for V(D)J recombination, e.g. XRCC4, DNA-ligase IV, Ku70, orKu80, Artemis, DNA-dependent protein kinase, catalytic subunit(DNA-PK_(cs)), and thus lack the ability to normally express ofendogenous antibody polypeptides.

Additional useful types of progenitor (pro) and precursor (pre) Blineage cells are early, immunoglobulin-null (Ig-null) EBV transformedhuman proB and preB cells (Kubagawa et al. PNAS 85, 875-879 (1988))(incorporated herein by reference in its entirety) that also express allthe required factors for expression, post-translational modification andsurface expression of exogenous antibodies (including CD79a and CD79b).

Other host cells of the B lineage can be used, that represent plasmacell differentiation stages of the B cell lineage, preferably, but notlimited to Ig-null myeloma cell lines, like Sp2/0, NSO, X63, Ag8653, andother myeloma and plasmacytoma cells, known in the art. Optionally,these cell lines may be stably transfected or stably geneticallymodified by other means than transfection, in order to over-express Bcell receptor components Ig-alpha (CD79a, or mb-1), and Ig-beta (CD79b,or B-29), in case optimal surface deposition of exogenously expressedantibodies is desired.

Other, non-lymphoid mammalian cells lines, including but not limited to,industry-standard antibody expression host cells, including, but notlimited to, CHO cells, Per.C6 cells, BHK cells and 293 cells may be usedas host cells for the method disclosed herein, and each of these cellsmay optionally also be stably transfected or stably genetically modifiedto over-express B cell receptor components Ig-alpha (CD79a, or mb-1),and Ig-beta (CD79b, or B-29), in case optimal surface deposition ofexogenously expressed antibodies is desired.

Essentially, any vertebrate host cell, which is transfectable, can beused for the method disclosed herein, which represents a major advantagein comparison to any viral expression systems, such as, but not limitedto vaccinia virus, retroviral, adenoviral, or sindbis virus expressionsystems, because the method disclosed herein exhibits no host cellrestriction due to virus tropism for certain species or cell types, andfurthermore can be used with all vertebrate cells, including humancells, at the lowest biosafety level, adding to its general utility.

Step (iii) of the method disclosed herein results in the stable geneticmodification of desired vertebrate host cells with the transfectedtransposable constructs of step (ii) by temporary, or transientexpression of a functional transposase enzyme, such that a stablepopulation of vertebrate host cells is generated that expresses diverselibraries of proteins encoded by said constructs.

A useful embodiment of step (iii) is to transiently introduce into thehost cells, preferably by co-transfection, as described above, avertebrate expression vector encoding a functional transposase enzymetogether with said at least one diverse transposable DNA library. It isto be understood that transient co-transfection or co-integration of atransposase expression vector can either be performed simultaneously, orshortly before or after the transfer of the transposable constructsand/or diverse transposable DNA libraries into the vertebrate hostcells, such that the transiently expressed transposase can optimally usethe transiently introduced transposable vectors of step (ii) for theintegration of the transposable DNA library into the vertebrate hostcell genome.

Another useful embodiment of step (iii) is to effect the stableintegration of the introduced transposable vectors and/or transposableDNA libraries of step (ii) by transiently expressing a functionaltransposase enzyme by means of an inducible expression system known inthe art, that is already stably integrated into the vertebrate host cellgenome. Such inducible and transient expression of a functionaltransposase may be achieved by e.g., tetracycline inducible(tet-on/tet-off) or tamoxifen-inducible promoter systems known in theart. In this case, only the one or more transposable vector or DNAlibrary needs to be introduced into the host cell genome, and the stabletransposition of the constructs and the stable expression of theproteins encoded by the one or more transposable vector or DNA libraryis effected by the transiently switched on expression of the functionaltransposase enzyme in the host cells.

Step (iv) of the method disclosed herein effects the isolation oftransposed vertebrate host cells expressing proteins with a desiredfunctionality or binding phenotype.

A preferred embodiment of step (iv) is to screen for and to isolate thetransposed host cells of step (iii) expressing desired proteins,including antibodies and fragments thereof, with target-binding assaysand by means of standard cell separation techniques, like magneticactivated cell sorting (MACS) or high-speed fluorescence activated cellsorting (FACS) known in the art. Especially, in a first enrichment stepof a specific population of transposed vertebrate host cells, wherelarge number of cells need to be processed, it is preferred to isolatetarget specific cells from a large number of non-specific cells byMACS-based techniques.

Particularly, for additional and iterative cell enrichment cycles, FACSenrichment is preferred, as potentially fewer numbers of cells need tobe processed, and because multi channel flow cytometry allows thesimultaneous enrichment of functionalities, including, but not limitedto, binding to a specific target of more than one species, or thespecific screening for particular epitopes using epitope-specificcompeting antibodies in the FACS screen.

If proteins, including antibodies and fragments thereof, are to bediscovered that interact with soluble binding partners, these bindingpartners are preferably labeled with specific labels or tags, such asbut not limited to biotin, myc, or HA-tags known in the art, that can bedetected by secondary reagents, e.g. but not limited to, streptavidin orantibodies, that themselves are labeled magnetically (for MACS basedcell enrichment) or with fluorochromes (for FACS based cell enrichment),so that the cell separation techniques can be applied.

If proteins, including antibodies and fragments thereof are to bediscovered against membrane bound proteins, which cannot easily beexpressed as soluble proteins, like e.g. but not limited to,tetraspannins, 7-transmembrane spanners (like G-coupled protein coupledreceptors), or ion-channels, these may be expressed in viral particles,or overexpressed in specific cell lines, which are then used forlabeling or panning methods known in the art, which can enrich thevertebrate host cells expressing the proteins from the transposedconstructs, including antibodies and fragments thereof.

Due to the stable genotype-phenotype coupling in the stably transposedvertebrate host cell population, a useful embodiment of step (v) is torepeat cell enrichment cycles for a desired functional or bindingphenotype, until a distinct population of cells is obtained that isassociated with a desired functional or binding phenotype. Optionally,individual cell clones can be isolated e.g., but not limited to, bysingle-cell sorting using flow cytometry technology, or by limitingdilution, in order to recover the transposed DNA information fromindividual cell clones that are coupled to a particular, desiredfunctional or binding phenotype.

For the identification of functional target-specific antibodies it isoften favorable to not only screen and to select for a particularbinding phenotype, but to additionally screen for additional functionalproperties of target specific antibodies, in particular antagonistic oragonistic effects in biological assay.

Therefore, it is desirable to be able to efficiently “switch” cellmembrane bound antibody expression to secreted antibody expression inthe vertebrate host cells with sufficient yields, in order to produceenough quantity of a particular antibody clone for functional assays.

In natural B lineage cells the switch from membrane bound to secretedantibody expression occurs via a mechanism of alternative splicing, inwhich in preB and B cells an alternative splice donor near the 3′ end ofthe last heavy chain constant region exon is preferentially spliced to asplice acceptor of a membrane anchor exon downstream of the heavy chainconstant regions exons. This way, an antibody heavy chain is produced inB cells with an extended C-terminal, membrane spanning domain, thatanchors the heavy chain and thereby the entire heavy-light chaincontaining antibody in the cell membrane. The C-terminal, membranespanning domain also interacts non-covalently with the membrane spanningcomponents Ig-alpha (CD79a or mb-1) and Ig-beta (CD79b or B29), whichlikely results in better membrane anchoring and higher surfaceimmunoglobulin expression in B lineage cells.

Once, a B cell differentiates further to the plasma cell stage, thealternative splicing does not occur anymore and the alternative splicedonor near the 3′ end of the last heavy chain constant region is nolonger recognized or utilized, and the mRNA template is terminateddownstream of the heavy chain constant region stop codon, and a heavychain of a secreted antibody is translated.

In order to exploit this natural mechanism of alternative splicing and“switching” from membrane bound to secreted expression of expressedantibodies, it is a useful embodiment of the method disclosed herein toconstruct the transposable vectors and diverse DNA libraries encodingproteins, including antibodies or fragments thereof, in such a way thatthe natural intron/exon structure of a constant antibody heavy chain,including the exons encoding the membrane spanning domains ismaintained. This embodiment represents a clear advantage againstretroviral expression systems, as the retroviral vector genome isalready spliced before it is packaged into a retroviral particle andstably transduced into the host cell genome.

Other viral vector systems may be restricted in the length of the DNAinsert that can be incorporated into the vectors, thereby precluding thecloning of larger genomic regions into such expression vectors andthereby preventing the exploitation of the natural “switching” frommembrane-bound to secreted antibody expression by alternative splicing.Certain transposons (e.g. Tol2, see FIG. 3), have been characterized tobe able to efficiently transpose more than 10 kb DNA fragments intovertebrate host cells without any loss in transposition efficiency(Kawakami Genome Biol. 8, Suppl I, S7 (2007)) (incorporated herein byreference in its entirety). Therefore, it is a useful embodiment of themethod disclosed herein to construct transposable expression vectorscomprising genomic exon/intron structures for better and properexpression and for the natural regulation switching from membrane boundto secreted antibody expression. The methods of the invention are usefulto transpose DNA fragments at least 5 kb, at least 6 kb, at least 7 kb,at least 8 kb, at least 9 kb, at least 10 kb in size into host cellgenomes.

The differentiation of earlier B lineage differentiation stage thatfavors membrane bound antibody expression, to a later, plasma cellstage, that favors secreted antibody expression can be induced by B celldifferentiation factors, such as, but not limited to, CD40/IL4triggering, or stimulation by mitogens, such as, but not limited to,lipopolysaccharide (LPS), or other polyclonal activators, Staph. aureusCowan (SAC) strain activators, and CpG nucleotides, or any combinationthereof.

Preferably, this differentiation is effected in transformed cells, inwhich the proliferation can artificially be inhibited, such that properB cell differentiation can again occur, as it has been described forA-MuLV transformed murine preB cells, in which the Abelson tyrosinekinase is specifically inhibited by the tyrosine inhibitor Gleevec(Muljo et al. Nat. Immunol 4, 31-37 (2003)) (incorporated herein byreference in its entirety). Therefore, it is a preferred embodiment toutilize Ig-null A-MuLV transformed murine preB cells for the method,which by treatment with Gleevec, can again differentiate to more matureB cell stages, including plasma cells, which then secrete sufficientamounts of secreted antibody for additional functional testing on thebasis of alternative splicing of genomic heavy chain expressionconstructs. It is a preferred embodiment of the method disclosed herein,to further improve such B-lineage cell differentiation by stableoverexpression of anti-apoptotic factors, known in the art, including,but not limited to, bcl-2 or bcl-x_(L).

After step (iv), the enrichment of transposed vertebrate host cells asdescribed above has been performed, optionally, additional cellenrichments according to the above-mentioned methods may be performed(step (v)), until cell populations, or individual cells are isolatedexpressing proteins, including antibodies and fragments thereof, withdesired functional and/or binding properties.

Step (vi) of the method disclosed herein is then performed in order toisolate the relevant coding information contained in the transposedvertebrate host cells, isolated for a desired functional and/or bindingproperty.

A useful embodiment of step (vi) for the isolation cloning andsequencing of the relevant coding information for a desired functionalor binding protein, including an antibody or antibody fragment thereof,contained in the isolated cells, is to utilize genomic or RT-PCRamplification with specific primer pairs for the relevant codinginformation comprised in the transposed DNA constructs, and to sequencethe genomic or RT-PCR amplicons either directly, or after sub-cloninginto sequencing vectors, known in the art, e.g., but not limited intoTA- or Gateway-cloning vectors.

Cloning and sequencing of the relevant coding information for a desiredfunctional or binding protein, including an antibody or antibodyfragment thereof, as described in the previous paragraph by genomic orRT-PCR amplification can also be performed with transposable IgH and IgLexpression vector, such that the binding protein coding region cannotonly be identified, but at the same time also be expressed upon stabletransposition into mammalian host cells as disclosed herein. For theexpression of secreted antibodies this would only require the use oftransposable Ig heavy chain expression vectors lacking the IgHtransmembrane spanning coding region.

Another useful embodiment of step (vi) is to subject the enriched cellpopulations of steps (iv) or (v), which exhibit a desired functional orbinding phenotype to next-generation (“deep”) sequencing (Reddy et al.Nat. Biotech. 28, 965-969 (2010)) (incorporated herein by reference inits entirety), in order to retrieve directly and in one step arepresentative set of several thousands of sequences for the codinginformation contained in the transposed DNA constructs. Based on abioinformatics analysis of the relative frequency of sequencesidentified from the enriched cell populations, it allows a predictionabout which sequences encoded a functional or binding protein, includingan antibody or fragment thereof (Reddy et al. Nat. Biotech. 28, 965-969(2010)) (incorporated herein by reference in its entirety).Statistically overrepresented sequences are then resynthesized andcloned into expression vector for expression as recombinant proteins,antibodies or fragments thereof, in order to characterize themfunctionally and for their binding properties. This method cansignificantly accelerate the identification of relevant sequences withina functionally and phenotypically enriched cell population, thatexpresses proteins with functional or target specific properties.

Yet another useful embodiment of the method disclosed herein is toutilize transposition-mediated vertebrate cell expression of proteins,including antibodies or fragments thereof, for the mutagenesis andoptimization of desired proteins, including the affinity optimization ofantibodies and fragments thereof.

This can be achieved by isolating the genes encoding the proteins,including antibody chains or fragments thereof, from transposedvertebrate cell populations enriched for a desired binding or functionalphenotype according to the methods disclosed in step (iv), such as butnot limited to, by genomic PCR or RT-PCR amplification undermutagenizing conditions, know in the art. The mutagenized sequences canthen be re-cloned into transposition vectors and then again betransposed into vertebrate host cells, in order to subject them toscreening according to the methods disclosed herein, for improvedfunctional or binding properties.

In one useful embodiment of this approach, specific primers are usedthat allow the PCR amplification under mutagenizing conditions ofcomplete transposed constructs, including the flanking ITRs.

By this method a mutagenized PCR amplicon containing a defined averagefrequency of random mutations is generated from the functionally orphenotypically selected transposed cells. Said PCR amplicon withcontrolled mutations (variations) of the original templates can nowdirectly be re-transposed into new vertebrate host cells, according topreferred embodiments disclosed in the methods applicable in step (ii).

The main advantage of this method over other approaches of geneticallymodifying vertebrate cells is, that with this technology notime-consuming re-cloning of the mutagenized PCR amplicons and timeconsuming quality control of the mutagenized sequences into expressionvectors is required, which is a mandatory requirement in all otherplasmid-based or viral expression systems, if a mutagenized sequenceshall be subjected to another round of screening.

Because transposition of DNA only requires the presence of ITRs flankingthe coding region of genes of interest, PCR-amplified mutagenized PCRamplicons can directly be re-introduced and re-transposed into novelvertebrate host cells for expression and screening for improvedproperties and/or affinity matured mutants.

Taken together, the methods disclosed herein, of utilizing TEs for thestable genetic modification of vertebrate host cells with transposableconstructs and/or diverse transposable DNA libraries encoding proteins,including antibodies and fragments thereof, offers unparalleledefficiency, flexibility, utility and speed for the discovery andoptimization of said proteins for optimal desired functional or bindingphenotypes.

EXAMPLES Example 1: Instruction for Cloning of Basic PiggyBacTransposable Light Chain Expression Vector for Human Antibody KappaLight Chains Compatible with the PiggyBac Transposase Enzyme

A basic transposable expression vector for human kappa light chains canbe generated by cloning of the ITRs from the PiggyBac transposon up anddownstream of a human immunoglobulin kappa light chain expressioncassette.

For this, as a first step, the minimal sequences for the up- anddownstream ITRs of the PiggyBac transposon can be derived from pXLBacII(published in U.S. Pat. No. 7,105,343) (incorporated herein by referencein its entirety) and can be gene synthesized with flanking restrictionenzyme sites for cloning into the mammalian expression vector pIRES-EGFP(PT3157-5, order #6064-1, Invitrogen-Life Technologies, Carlsbad,Calif., USA)

The upstream PiggyBac ITR sequence with the 5′ terminal repeat has to begene synthesized with flanking MunI restriction enzyme sequence,compatible with a unique MunI restriction enzyme site in pIRES-EGFP, andadditional four random nucleotides (in lowercase letters) allowingproper restriction enzyme digestion. This sequence is provided in SEQ IDNO:1.

The downstream PiggyBac ITR sequence with the 3′ terminal repeat has tobe gene synthesized with flanking XhoI restriction enzyme sequencecompatible with a unique XhoI restriction enzyme site in pIRES-EGFP, andadditional four random nucleotides allowing proper restriction enzymedigestion. This sequence is provided in SEQ ID NO:2. Upon MunIrestriction enzyme digestion of the gene synthesized SEQ ID NO:1, theDNA fragment can be ligated into MunI linearized pIRES-EGFP, generatingpIRES-EGFP-TR1 according to standard methods, known in the art. Theproper orientation of the insert can be verified by diagnosticrestriction enzyme digestion, and/or by DNA sequencing of the clonedconstruct (FIG. 5a ).

In a next step gene synthesized and XhoI digested DNA fragment SEQ IDNO:2, can be ligated into XhoI linearized pIRES-EGFP-T1 (FIG. 5a ) bystandard methods known in the art, in order to generate pIRES-EGFP-T1T2,containing both PiggyBac ITRs up and downstream of the IRES-EGFPexpression cassette (FIG. 5b ). The proper orientation of the insert canbe verified by diagnostic restriction enzyme digestion, and/or by DNAsequencing of the cloned construct (FIG. 5b ).

For the cloning of a human immunoglobulin kappa light chain into thevector pIRES-EGFP-T1T2, the human Ig kappa light chain from humananti-TNF-alpha specific antibody D2E7 can be synthesized, which can beretrieved from European patent application EP 1 285 930 A2 (incorporatedherein by reference in its entirety).

The coding region for human Ig kappa light chain of human anti-TNF-alphaspecific antibody D2E7, in which the V region of D2E7 is fused in frameto a Vk1-27 leader sequence (Genbank entry: X63398.1, which is theclosest germ-line gene V-kappa family member V-kappa of D2E7), and tothe human kappa constant region (Genbank entry: J00241) has thefollowing nucleotide sequence, which is provided in SEQ ID NO:3.

The nucleotide sequence of SEQ ID NO:3 translates in the amino acidsequence SEQ ID NO:4. The DNA fragment SEQ ID NO:3 encoding the D2E7 Igkappa light chain can be gene synthesized and directly ligated byblunt-ended ligation into the unique EcoRV restriction enzyme site(which is also a blunt cutter), by methods know in the art, resulting inconstruct pIRES-EGFP-T1T2-IgL (FIG. 5b )

SEQ ID NO:3 has been engineered to contain a unique Eco47III restrictionenzyme site in between the V-kappa and the C-kappa coding regions(highlighted in boldface and underlined), which allows the replacementof V-kappa regions in this construct against other V-kappa regions orV-kappa libraries, using a unique restriction enzyme upstream of V-kappacoding region in the construct, together with Eco47III. The properorientation of the kappa light chain insert can be verified bydiagnostic restriction enzyme digestion, and/or by DNA sequencing of thecloned construct (FIG. 5b ).

The entire sequence for the transposable human antibody kappa lightchain vector pIRES-EGFP-T1T2-IgL is provided in sequence SEQ ID NO:5.

Sequences Referred to in this Example 1

SEQ ID NO:1 (327 bp long PiggyBac 5′-ITR sequence. The MunI restrictionenzyme sites at each end are underlined and typed in boldface print, therandom nucleotide additions at the termini are printed in lowercase)

Seq-ID2 (264 bp long PiggyBac 3′-ITR sequence. The XhoI restrictionenzyme sites at each end are underlined and typed in boldface print, therandom nucleotide additions at the termini are printed in lowercase)

SEQ ID NO:3 (711 bp long Ig-kappaLC coding region ofanti-TNF-alpha-specific mAb D2E7)

SEQ ID NO:4 (236 amino acids long sequence of anti-TNF-alpha-specificmAb D2E7)

SEQ ID NO:5 (6436 bp long DNA sequence of PiggyBac transposableIg-kappa-LC expression vector pIRES-EGFP-T1T2-IgL)

Example 2: Instruction for Cloning of a Basic PiggyBac TransposableHeavy Chain Expression Vector for Membrane Spanning Human AntibodyGamma1 Heavy Chains

In order to clone a transposable Ig heavy chain expression vector, thekappa light chain ORF from pIRES-EGFP-T1T2-IgL (SEQ ID NO:5) needs to beexchanged with an ORF encoding a fully human IgG1 heavy chain codingregion.

For the replacement of the human kappa light chain in vectorpIRES-EGFP-T1T2-IgL by a human immunoglobulin gamma-1 heavy chain, theV_(H) region of antibody D2E7, which is specific for human TNF-alpha(see: EP 1 285 930 A2) (incorporated herein by reference in itsentirety) can be synthesized. For this, a leader sequence of a closegerm-line V_(H)3-region family member is fused in frame to the V_(H)region of antibody D2E7, which then is fused in frame to the codingregion of a human gamma1 constant region (Genbank: J00228) including themembrane spanning exons (Genbank: X52847). In order to be able toreplace the human Ig kappa light chain from pIRES-EGFP-T1T2-IgL, uniqueClaI and NotI restriction enzyme sites need to be present at the 5′ andthe 3′ end of the sequence (underlined), respectively. Additionally,four nucleotides flanking the restriction enzyme sites (highlighted inlowercase letters at the ends of the sequence) allow proper restrictionenzyme digestion of the gene-synthesized DNA fragment and ligation intothe ClaI-Nod linearized pIRES-EGFP-T1T2-IgL backbone, according tostandard methods. The sequence that needs to be gene synthesized isprovided in SEQ ID NO:6.

From the start codon in position 11 of SEQ ID NO:6 (highlighted inboldface print), this nucleotide sequence translates to the human IgG1heavy chain of anti-TNF-alpha specific clone D2E7 (see: EP 1 285 930 A2)(incorporated herein by reference in its entirety), but including thehuman gamma1 transmembrane exons M1 and M2. The protein translation ofSEQ ID NO:6 is provided in SEQ ID NO:7. The DNA fragment SEQ ID NO:6encoding the D2E7 Ig gamma-1 heavy chain can then be double-digested byClaI and NotI restriction enzymes and directionally ligated into ClaIand Nod linearized pIRES-EGFP-T1T2-IgL, resulting in constructpIRES-EGFP-T1T2-IgH (FIG. 6)

SEQ ID NO:6 has also been engineered to contain a unique Eco47IIIrestriction enzyme site in between the V-heavy variable and the C-gamma1constant coding regions (highlighted in boldface and underlined in SEQID NO:7), which allows the replacement of V-heavy regions in thisconstruct against other V-heavy regions or V-heavy libraries, using aunique restriction enzyme upstream of V-heavy coding region in theconstruct, together with Eco47III. The correct ligation of the insertcan be verified by diagnostic restriction enzyme digestion, and/or byDNA sequencing of the cloned construct (FIG. 6).

The entire sequence for the transposable human antibody gamma-1 heavychain vector pIRES-EGFP-T1T2-IgH is provided in sequence SEQ ID NO:8

Examples 1 and 2 provide instructions for the cloning of basic PiggyBactransposable expression vectors for human antibody kappa light and humangamma-1 heavy chains (membrane bound form) and therefore forfull-length, membrane bound human IgG1, that can be utilized for thereduction to practice of the invention.

Sequences Referred to in this Example 2

SEQ ID NO:6 (1642 bp long DNA fragment containing the coding region formembrane bound Ig-gamma1-HC of anti-TNF-alpha-specific mAb D2E7)

SEQ ID NO:7 (539 amino acids long sequence of membrane boundIg-gamma1-HC of anti-TNFalpha antibody

SEQ ID NO:8 (7341 bp long DNA sequence of PiggyBac transposable humanIg-gamma1-membrane-HC expression vector pIRES-EGFP-T1T2-IgH)

Example 3: Instructions for Cloning of Basic Transposable Light ChainExpression Vector for Human Antibody Kappa Light Chains Compatible withthe Sleeping Beauty Transposase Enzyme

In order to transpose human immunoglobulin heavy and light chainexpression vectors contained in a transposable vector independently intohost cells, a transposable immunoglobulin light chain construct withdifferent inverted terminal repeat (ITR) sequences can be constructedthat are recognized by the Sleeping Beauty transposase.

For this, the human Ig-kappa light chain expression vectorpIRES-EGFP-T1T2-IgL (SEQ ID NO:5) of example 1 can be used to replacethe 5′ and 3′ ITRs of the PiggyBac transposon system, contained in thisvector, with the 5′ and 3′ ITRs of the Sleeping Beauty transposonsystem. The sequences for the Sleeping Beauty 5′ITR and 3′ITR,recognized and functional with the Sleeping Beauty transposase, can beretrieved from patent document US7160682B1/US2003154500A1.

The upstream Sleeping beauty ITR sequence with the 5′ terminal repeathas to be gene synthesized with flanking MunI restriction enzymesequences, allowing the replacement of the MunI flanked PiggyBac 5′ITRin construct pIRES-EGFP-T1T2-IgL (SEQ ID NO:5) of example 1 by theSleeping Beauty 5′ITR sequence. This sequence is provided as SEQ IDNO:14 below, at the end of this Example.

The downstream Sleeping beauty ITR sequence with the 3′ terminal repeat(also published in U.S. Pat. No. 7,160,682B1/US2003154500A1) has to begene synthesized with flanking XhoI restriction enzyme sequences,allowing the replacement of the XhoI flanked PiggyBac 3′ITR in constructpIRES-EGFP-T1T2-IgL (SEQ ID NO:5) of example 1 by the Sleeping beauty3′ITR sequence. This sequence is as provided in SEQ ID NO:15 below, atthe end of this Example (XhoI restriction enzyme sites are highlightedin boldface print and 4 additional flanking random nucleotides, allowingproper restriction enzyme digestion of the gene synthesized fragment,are indicated in lowercase letters):

In a first step, the MunI-flanked PiggyBac 5′ITR of constructpIRES-EGFP-T1T2-IgL (SEQ ID NO:5) has to be replaced by the SleepingBeauty 5′ITR by digesting pIRES-EGFP-T1T2-IgL (SEQ ID NO:5) with MunIrestriction enzyme and by ligating the MunI digested gene-synthesizedfragment from SEQ ID NO:14 into the MunI linearized vector backbone ofpIRES-EGFP-T1T2-IgL (SEQ ID NO:5). The correct orientation of SleepingBeauty 5′ITR can be checked by diagnostic restriction enzyme digestionsand/or DNA sequencing. The resulting plasmid is calledpIRES-EGFP-sbT1-pbT2-IgL (FIG. 8).

In a second step, the XhoI-flanked PiggyBac 3′ITR of construct stillcontained in pIRES-EGFP-sbT1-pbT2-IgL has to be replaced by the SleepingBeauty 3′ITR by digesting pIRES-EGFP-sbT1-pbT2-IgL with XhoI restrictionenzyme and by ligating the XhoI digested gene-synthesized fragment fromSEQ ID NO:15 into the XhoI linearized vector backbone ofpIRES-EGFP-sbT1-pbT2-IgL. The correct orientation of Sleeping Beauty3′ITR can be checked by diagnostic restriction enzyme digestions and/orDNA sequencing. The resulting plasmid is called pIRES-EGFP-sbT1T2-IgL(FIG. 8).

The entire sequence of the Ig-kappa LC expression vectorpIRES-EGFP-sbT1T2-IgL transposable by the Sleeping Beauty transposase isprovided in SEQ ID NO:16 below, at the end of this Example.

Sequences Referred to in this Example 3

SEQ ID NO:14 (246 bp long DNA fragment containing the 5′ITR of theSleeping Beauty transposon system. Flanking MunI restriction enzymesites are printed in boldface and underlined)

SEQ ID NO:15 (248 bp long DNA fragment containing the 3′ITR of theSleeping Beauty transposon system)

SEQ ID NO:16 (6339 bp long DNA sequence of Sleeping Beauty transposableIg-kappa-LC expression vector pIRES-EGFP-sbT1T2-IgL)

Example 4: Cloning of PiggyBac and Sleeping Beauty Transposable Vectorsfor Membrane Bound Human IgG₁

In addition to the cloning instructions for basic PiggyBac transposableIgH and IgL expression vectors provided in Examples 1 and 2, and theconstruction of a basic Sleeping Beauty transposable IgL expressionvector provided in Example 3, additionally cloning of improved PiggyBacand Sleeping Beauty transposable IgH and IgL expression vectors for achimeric anti-humanCD30 mAb and for a humanized anti-humanCD19 mAb hasbeen performed, in order to reduce the invention to practice.

For this, in a first step, the following two gene fragments have beensynthesized (commissioned to Genscript, Piscataway, N.J., USA):

1.) A 4975 bp DNA fragment containing an expression cassette, in whichthe expression of a human membrane bound IgG₁ heavy chain is driven bythe EF1-alpha promoter (basepairs 1-1335 of Clontech expression vectorpEF1-alpha-IRES, Cat-No. #631970), and in which the expression of Igchains is linked to EGFP expression via an internal ribosomal entry site(IRES). The DNA sequences for the IRES and EGFP regions were derivedfrom pIRES-EGFP (basepairs 1299-1884 and 1905-2621, respectively, ofClontech expression vector pIRES-EGFP (Cat.-No. #6064-1, LifeTechnologies). In addition, the synthesized DNA fragment contained achimeric intron positioned in between the Ig constant coding region andthe IRES sequence, whose sequence was derived from pCI mammalianexpression vector (basepairs 857-989. od Promega, Cat.-No. #E1731). Atthe 3′ end of the expression cassette the synthesized fragment containeda bovine growth hormone polyadenylation signal (BGH-polyA), whosesequence was derived from pCDNA3.1-hygro(+) expression vector (basepairs1021-1235 of Invitrogen-Life Technologies, Cat.-No. #V870-20). Theexpression cassette was flanked up- and downstream by PiggyBactransposon ITRs already disclosed in SEQ ID NO:1 and SEQ ID NO:2 furtherabove.

A map of the elements and their arrangement in the gene-synthesized DNAfragment is provided in FIG. 10, including additionally added uniquerestriction enzyme sites that can be used to excise or to replace any ofthe functional elements of the expression cassette.

The sequence of the 4975 bp long gene-synthesized fragment is providedas SEQ ID NO:20 below, at the end of this Example.

It shall be noted here that the gene synthesized expression cassette forhuman IgH chains provided in SEQ ID NO:20, on purpose, did not yetcontain the coding region for a V_(H) domain, such that the constructcan be used for the insertion of any desired V_(H) coding region and/orV_(H) coding gene library using unique restriction enzyme sites Nod andNheI. This construct therefore is designated “empty” Ig-gamma1-HCexpression cassette.

2.) In order to provide a plasmid backbone for the transposableexpression cassette of SEQ ID NO:20, a 2774 bp long DNA fragment hadbeen gene synthesized (performed by Genscript, Piscataway, N.J., USA)that contained a bacterial ColE1 on and an ampicillin resistance gene.The sequence information for these plasmid backbone components werederived from the plasmid backbone of expression vector pCI (Promega,Cat.-No. #E1731). The synthetic gene fragment additionally contained 5′and 3′ ITRs of the Sleeping Beauty transposon, already disclosed in SEQID NO:14 and SEQ ID NO:15, respectively. This fragment needed to becircularized and could be propagated in E. coli as an autonomousplasmid, due to the presence of the ColE1 on and the ampicillinresistance gene.

A map of the elements and their arrangement in the gene-synthesized DNAfragment is provided in FIG. 10, including position of additionallyadded unique restriction enzyme sites that can be used to excise or toreplace any of the functional elements of the expression vector.

The sequence of the 2774 bp long gene-synthesized fragment is providedas SEQ ID NO:21 below at the end of this Example:

These two gene fragments allowed the construction of both PiggyBac andSleeping Beauty transposable vectors by ligating fragments from thesevectors, upon digestion with different restriction enzymes, followed byligation, as follows:

The PiggyBac transposable vector was cloned by ligating EcoRI-ClaIfragments from SEQ ID NO:20 and SEQ ID NO:21, such that the resultingconstruct contains the entire PiggyBac ITR-flanked expression cassetteof SEQ ID NO:20, and the ColE1-amp containing backbone without theSleeping Beauty ITRs of Sq-ID21. Conversely, the ligation of XbaI-MluIfragments from SEQ ID NO:20 and SEQ ID NO:21 resulted in the ligation ofthe expression cassette without the PiggyBac ITRs into the linearizedplasmid backbone of SEQ ID NO:21 still containing the Sleeping BeautyITRs. Miniprep plasmids resulting from the two ligations were analyzedby diagnostic restriction enzyme digestions using a mixture ofXhoI-NheI-BamHI and in addition with PvuI restriction enzymes, in orderto identify correctly ligated plasmids. One selected DNA clone of eachligation was retransformed into E. coli to generate a DNA maxiprep,which was verified by DNA sequencing using sequencing primers allowingsequencing of the entire plasmid sequence.

The entire sequences of PiggyBac and Sleeping beauty transposablevectors (containing the “empty” human gamma1-HC expression cassette)generated as described above and verified by DNA sequencing is providedas SEQ ID NO:22 (PiggyBac transposable vector) and SEQ ID NO:23(Sleeping Beauty transposable vector) below, at the end of this Example.

V_(H) and V_(L) coding regions of chimeric anti-human CD30 antibodybrentuximab (clone Ac10) could be retrieved from sequences 1 and 9 ofpatent application US2008213289A1, and are provided below as SEQ IDNO:24 and SEQ ID NO:25, respectively.

V_(H) and V_(L) coding regions of humanized anti-human CD19 antibodyhBU12 were retrieved from patent document U.S. Pat. No. 8,242,252 B2 assequence variants HF and LG, respectively, and are provided in SEQ IDNO:26 and SEQ ID NO:27 further below, at the end of the Example.

In order to allow construction of final PiggyBac and Sleeping Beautytransposable anti-CD30 and anti-CD19 IgHC expression vectors, the DNAfragments for the V_(H) domains were designed to have flanking NheI andNod restriction enzyme sites. The nucleotide sequence encoding the V_(H)of anti-CD30 antibody brentuximab (clone Ac10) has additionally beenmodified to also contain a leader sequence for mammalian cellexpression. The DNA sequences of the NotI-NheI fragments encoding theV_(H) of anti-CD30 and anti-CD19 mAbs are provided in SEQ ID NO:28 andSEQ ID NO:29 at the end of this Example. The DNA fragments had been genesynthesized by GeneArt, Regensburg, Germany (NotI and NheI sites areunderlined).

In order to generate anti-CD30 and anti-CD19 IgH chain expressionvectors that are transposable with either PiggyBac or Sleeping Beautytransposase, the NotI-NheI digested fragments of SEQ ID NO:28 SEQ IDNO:29 were ligated into NotI-NheI linearized vectors disclosed in SEQ IDNO:22 or SeqID23, respectively. This resulted in the generation of fourvectors containing a fully functional heavy chain (HC) of anti-CD30 mAbbrentuximab (clone Ac10) and of anti-CD19 mAb hBU12 and the constructswere designated: pPB-EGFP-HC-Ac10, pPB-EGFP-HC-hBU12, pSB-EGFP-HC-Ac10,and pSB-EGFP-HC-hBU12 and their vector maps are provided in FIG. 11.These vectors have specifically been designed to allow surfaceexpression of the heavy chains, and, upon co-expression of light chains,surface IgG expression. However, simple omission of the coding region offor the membrane spanning region of the Ig heavy chains would result intransposable expression vectors for secreted IgG.

In order to generate anti-CD30 and anti-CD19 IgL chain expressionvectors that are transposable with either PiggyBac or Sleeping Beautytransposase, the IgH constant region genes from the vectors disclosed inSEQ ID NO:22 and SEQ ID NO:23 needed to be replaced with IgL chaincoding regions of anti-CD30 and anti-CD19 antibodies. This was achievedby gene synthesizing gene fragments containing the V_(L) coding regionsas disclosed in SEQ ID NO:25 and SEQ ID NO:27 fused in-frame to a humanconstant kappa light chain coding region, with a leader sequence at the5′ end and flanked by NotI-BstBI cloning sites that allow the ligationof the NotI-BstBI digested fragment into NotI-BstBI linearized vectorsdisclosed in SEQ ID NO:22 and SEQ ID NO:23, thereby replacing the IgHconstant coding region of SEQ ID NO:22 and SEQ ID NO:23 with the IgLcoding regions of anti-CD30 mAb Ac10 and anti-CD19 mAb hBU12.

The gene-fragments containing the IgL coding regions of anti-CD30 mAbAc10 and anti-CD19 mAb hBU12, with leader sequence and flanked byNotI-BstBI cloning sites is disclosed in SEQ ID NO:30 and SEQ ID NO:31below, at the end of the Example. The gene synthesis of these DNAfragments was performed by Genscript (Piscataway, N.J., USA).

In order to generate anti-CD30 and anti-CD19 IgL chain expressionvectors that are transposable with either PiggyBac or Sleeping Beautytransposase, NotI-BstBI digested fragments of SEQ ID NO:30 and SEQ IDNO:31 had been ligated into NotI-BstBI linearized vectors disclosed inSEQ ID NO:22 or SeqID23. The resulting four vectors were called:pPB-EGFP-LC-Ac10, pPB-EGFP-LC-hBU12, pSB-EGFP-LC-Ac10, andpSB-EGFP-LC-hBU12 and their vector maps are provided in FIG. 11.

Complete sequences of the PiggyBac and Sleeping beauty anti-CD30 andanti-CD19 IgH and IgL constructs (eight combinations) are provided inSEQ ID NO:32 (pPB-EGFP-HC-Ac10), SEQ ID NO:33 (pPB-EGFP-HC-hBU12), SEQID NO:34 (pSB-EGFP-HC-Ac10), SEQ ID NO:35 (pSB-EGFP-HC-hBU12), and inSEQ ID NO:36 (pPB-EGFP-LC-Ac10), SEQ ID NO:37 (pPB-EGFP-LC-hBU12), SEQID NO:38 pSB-EGFP-LC-Ac10), and SEQ ID NO:39 (pSB-EGFP-LC-hBU12) below,at the end of this Example

Sequences Referred to in this Example

SEQ ID NO:20 (4975 bp long DNA sequence containing a PiggyBacITR-flanked expression cassette for membrane spanning human Ig-gamma1heavy chains)

SEQ ID NO:21 (2774 bp long DNA sequence containing vector backbonecomponents ColE1 and ampicillin resistance flanked by 5′ and 3′ ITRs ofSleeping Beauty)

SEQ ID NO:22 (7242 bp long sequence of PiggyBac transposable “empty”human gamma1-HC vector)

SEQ ID NO:23 (7146 bp long sequence of Sleeping Beauty transposable“empty” human gamma1-HC vector)

SeqID-24 (351 bp long V_(H) coding region of anti-human CD30 antibodybrentuximab)

SEQ ID NO:25 (333 bp long V_(L) coding region of anti-human CD30antibody brentuximab)

SEQ ID NO:26 (417 bp long V_(H) coding region of anti-human CD19 mAbhuB12, including leader)

SEQ ID NO:27 (375 bp long V_(L) coding region of anti-human CD19 mAbhuB12, including leader)

SEQ ID NO:28 (423 bp long DNA fragment, containing NotI-NheI-flankedV_(H) coding region of the V_(H) domain of anti-human CD30 mAbbrentuximab)

SEQ ID NO:29 (432 bp long DNA fragment, containing NotI-NheI-flankedV_(H) coding region of the V_(H) domain of anti-human CD19 mAb hBU12)

SEQ ID NO:30 (733 bp long DNA fragment containing IgL coding region ofanti-CD30 mAb Ac10 and flanked by NotI and BstBI restriction enzymesites)

SEQ ID NO:31 (718 bp long DNA fragment containing IgL coding region ofanti-CD19 mAb hBU12 and flanked by NotI and BstBI restriction enzymesites)

SEQ ID NO:32 (7645 bp sequence of pPB-EGFP-HC-Ac10

SEQ ID NO:33 (7654 bp sequence of pPB-EGFP-HC-hBU12)

SEQ ID NO:34 (7549 bp sequence of pSB-EGFP-HC-Ac10)

SEQ ID NO:35 (7558 bp sequence of pSB-EGFP-HC-hBU12)

SEQ ID NO:36 (6742 bp long sequence of pPB-EGFP-LC-Ac10)

SEQ ID NO:37 (6727 bp long sequence of pPB-EGFP-LC-hBU12)

SEQ ID NO:38 (6646 bp long sequence of pSB-EGFP-LC-Ac10)

SEQ ID NO:39 (6631 bp long sequence of pSB-EGFP-LC-hBU12)

Example 5: Instructions for Cloning of a PiggyBac Transposase ExpressionVector

The ORF of functional PiggyBac transposase enzyme can be retrieved fromU.S. Pat. No. 7,105,343 B1 (incorporated herein by reference in itsentirety) and is provided in SEQ ID NO:11 below, at the end of thisExample. The DNA sequence of SEQ ID NO:11 translates into the amino acidSEQ ID NO:12 also provided at the end of this Example.

In order to generate a vertebrate cell expression vector for thePiggyBac transposase enzyme, this ORF can be gene synthesized and clonedas a blunt ended DNA into the unique, blunt-cutting restriction enzymesite EcoRV in the standard vertebrate cell expression vectorpCDNA3.1-hygro(+) (catalogue # V870-20, Invitrogen, Carlsbad, Calif.,USA), by methods know in the art. The correct ligation of the PiggyBacORF, relative to the pCDNA3 promoter can be verified by diagnosticrestriction enzyme digestion, and/or by DNA sequencing of the clonedPiggyBac expression construct pCDNA3.1-hygro(+)-PB (FIG. 7). Theconstruction of a PiggyBac expression vector was performed as describedherein and the vector design was verified by diagnostic restrictionenzyme digestion, and DNA sequencing.

The sequence of the PiggyBac expression construct pCDNA3.1-hygro(+)-PBis provided as SEQ ID NO: 13, below at the end of this Example

Sequences Referred to in this Example 5

SEQ ID NO:11 (ORF of PiggyBac transposase)

SEQ ID NO:12 (amino acid sequence of PiggyBac transposase)

SEQ ID NO:13 (pCDNA3.1-hygro(+)-PiggyBac expression vector)

Example 6: Instructions for Cloning of a Sleeping Beauty TransposaseExpression Vector

The open reading frame (ORF) of the Sleeping Beauty transposase enzymecan be found in patent reference U.S. Pat. No.7,160,682B1/US2003154500A1. The sequence is provided in SEQ ID NO:17,below at the end of this Example. This DNA sequence of SEQ ID NO:17translates into the amino acid sequence of SEQ ID NO:18, also providedat the end of this Example, further below.

In order to generate a vertebrate cell expression vector for theSleeping Beauty transposase enzyme, this ORF can be gene synthesized andcloned as a blunt ended DNA into the unique, blunt-cutting restrictionenzyme site EcoRV in the standard vertebrate cell expression vectorpCDNA3.1-hygro(+) (catalogue # V870-20, Invitrogen, Carlsbad, Calif.,USA), by methods know in the art. The correct ligation of the SleepingBeauty ORF, relative to the pCDNA3 promoter can be verified bydiagnostic restriction enzyme digestion, and/or by DNA sequencing of thecloned Sleeping Beauty expression construct pCDNA3.1-hygro(+)-SB (FIG.9).

The sequence of the Sleeping Beauty expression constructpCDNA3.1-hygro(+)-SB is provided in SEQ ID NO:19, below, at the end ofthis Example.

The construction of a Sleeping Beauty expression vector was performed asdescribed herein and the vector design was verified by diagnosticrestriction enzyme digestion, and DNA sequencing. The coding regions forPiggyBac and Sleeping Beauty transposase enzymes had been genesynthesized by Genscript, Piscataway, N.J. With the eight differenttransposable IgH and IgL expression vectors for PiggyBac and SleepingBeauty transposases, and the pCDNA3.1-hygro(+) expression vectors forPiggyBac and Sleeping Beauty transposase enzymes, all vectors have beengenerated that allow the expression of anti-CD30 and anti-CD19antibodies on the cell surface of mammalian cells.

Sequences Referred to in this Example 6

SEQ ID NO:17 (ORF of Sleeping Beauty transposase enzyme)

SEQ ID NO:18 (amino acid sequence of Sleeping Beauty transposase)

SEQ ID NO:19 (DNA sequence of Sleeping Beauty expression vectorpCDNA3.1-hygro(+)-SB)

Example 7: Generation of Murine preB Cells Stably Expressing MembraneBound Human IgG from Stably Transposed Expression Vectors

In order to demonstrate stable expression of human IgG antibodies inmammalian cells, transposable human IgH and IgL expression constructshave been transfected into Abelson murine leukemia virus (A-MuLV)transformed proB cell line 63-12, originally derived from RAG-2deficient mice (Shinkai et al. (1992) Cell 68, 855-867) and thereforeunable to initiate V(D)J recombination. This host cell line represents aB cell lineage lymphocyte cell type that expresses all cellularcomponents for optimal membrane bound antibody expression, including theB cell receptor co-factors Ig-alpha (CD79a or mb-1) and Ig-beta (CD79bor B29) that interact with the transmembrane spanning amino acids ofmembrane bound immunoglobulin. Therefore, these cells optimally anchorIgG molecules with a trans-membrane spanning region in the cell surfacemembrane. 63-12 cells were grown in static culture in suspension usingIMDM medium supplemented with 2% FCS, 0.03% Primatone™ RL-UF (SheffieldBioscience), 2 mM L-glutamine, 50 μM 2-mercaptoethanol, at 37° C. in ahumidified incubator and a 10% CO₂ atmosphere. For the co-transfectionof the transposable IgH and IgL expression vectors (Example 4) with atransposon expression vector (Examples 5 or 6), the cells were passaged24 hours prior to transfection and seeded at a density of 5×10⁵cells/ml, in order to allow the cells to enter into log-phase growthuntil the time-point of transfection.

At the day of transfection, 63-12 cells were harvested by centrifugationand resuspended in RPMI 1640 medium without any supplements or serum ata density of 5×10⁶ cells/ml. 400 μl of this cell suspension(corresponding to 2×10⁶ cells) were transferred into 0.4 cmelectroporation cuvettes (BioRad order #165-2081) and mixed with 400 μlof RPMI 1640 medium containing the desired plasmid DNA (or a mixture ofplasmids). Cells were then transfected using a BioRad Gene Pulser II at950 μF/300V settings and incubated for 5 min at room temperature after asingle electroporation pulse. After this, the cells were transferredinto 5 ml IMDM-based growth medium and the cells were centrifuged once,in order to remove cell debris and DNA from the electroporation, beforethe cells were transferred into IMDM-based growth medium for recoveryand expression of proteins from transfected plasmids.

The electroporation settings have been determined as the most optimaltransfection conditions for A-MuLV transformed proB cell line 63-12 thatroutinely resulted in transient transfection efficiencies rangingbetween 30-40%. The result of such a transfection by electroporation isdocumented in the FACS analyses depicted in FIG. 12, where thetransfection controls, two days post transfection, are depicted on theleft column panels. The negative control (labeled NC), that wasmock-electroporated without DNA, as expected, does not show any greenfluorescent cells, whereas the transfection control that was transfectedwith 15 μg pEGFP-N3 plasmid (Clontech, order #6080-1), showed that 38.8%of the cells were transiently transfected, as detected by cellsexpressing enhanced green fluorescent protein (see cell in lower rightquadrant). As expected, the transfection controls do not show anyIg-kappa signal, because none of the transfection controls wastransfected with an Ig-expression construct.

For transposition of IgH and IgL expression vectors, 63-12 cells werealso transfected by electroporation with a mix of 5 μg each of atransposable IgH expression vector, 5 μg of a transposable IgLexpression vector, and 5 μg of an expression vector allowing expressionof a transposase mediating the transposition of the IgH and IgLexpression vector. The result of this transfection is also shown in FIG.12.

Expression of human IgG on the surface of the cells was detected by abiotinylated anti-human kappa light chain specific antibody (Affymetrix,ebioscience, order #13-9970-82) detected withstreptavidin-allophycocyanin (strep-APC) (Affymetrix, ebioscience, order#17-4317-82), and is shown on the Y-axis of the FACS dot-plots. As canbe seen in FIG. 10, the measurements depicted in the second column fromleft show the analysis of cells transfected with IgH+IgL+transposaseexpression vectors two days after electroporation (labeled “d2 postTF”). The FACS analysis after two days of transfection showed thatbetween 1.8% and 2.8% of the cells express human IgH+IgL on the cellsurface, because IgL expression on the cell surface can only bedetected, if IgH chains are co-expressed in the cells, such that acomplete IgG can be expressed on the surface of the cells.

From this data it can be inferred that if ca. 38% of the cells aretransiently transfected, ca. 5-7.5% of these cells have beenco-transfected with both IgH and IgL expression vectors. From thisexperiment it is concluded that the transposable IgH and IgL expressionconstructs allow high-level expression of human IgG on the surface ofmurine A-MuLV transformed proB cells, which is comparable to surface IgGsignals obtained by staining of human peripheral B lymphocytes with thesame antibody staining reagents (data not shown).

As expected the cells showing IgG expression also displayed EGFPexpression, because the EGFP expression was transcriptionally coupled toIgH or IgL expression via IRES sequences. However, the EGFP expressionwas significantly lower, as compared to the EGFP expression from thepEGFP-N3 control plasmid, which is expected, as the EGFP expression inpEGFP-N3 is directly driven by a strong constitutive promoter, whereasEGFP expression in the transposable IgH and IgL expression vectors iseffected by transcriptional coupling to the IgH and IgL coding regionusing an internal ribosomal entry site (IRES). Nevertheless, asexpected, cells displaying higher IgG expression also displayed higherEGFP signals (leading to a slightly diagonal Ig-kappa⁺/EGFP⁺population), which clearly demonstrates, that both expression levels arecoupled.

When cells were analyzed without cell sorting after one week oftransfection, the EGFP signal in pEGFP-N3 control transfections was nolonger detectable (data not shown), showing that the cells do not stablyintegrate expression constructs at any significant frequency. Incontrast, a low ca. 1-2% IgG-EGFP double-positive population of cellswas maintained in cells that have been co-transfected with transposableIgH&IgL vectors together with a transposase expression vector, alreadyindicating that ca. 3-6% of the cell transiently transfected cellsstably integrate simultaneously the transposable IgH and IgL expressionvectors into their genome (data not shown).

In order to enrich for these stably transposed cells, Ig-kappa lightchain and EGFP double positive cells have been FACS sorted at day 2 posttransfection, as indicated by the sorting gates (black rectangles in thesecond left column FACS dot plots). Each 5,000 cells falling into thisgate have been sorted from the PiggyBac transpositions with IgH&IgL ofanti-CD30 mAb Ac10 (top row), and of anti-CD19 mAb hBU12 (middle row),and 3,000 cells have been sorted from the Sleeping Beauty transpositionwith IgH&IgL of anti-CD30 mAb Ac10 (bottom row), as indicated in FIG.12.

The FACS-sorted cells were expanded for one week (representing day 9post transfection), and were re-analyzed again for surface IgGexpression by detection of IgG with an anti-kappa light chain antibody,as described above. As can be seen in FIG. 10 (second column from theright), over 30%, 50% and 5% of the one-time sorted cells stablyexpressed IgG on the cell surface, while these cells, as expected, werealso EGFP-positive. This demonstrates that a significant percentage oftransiently IgH & IgL co-transfected cells stably maintain IgGexpression. The PiggyBac mediated transpositions in this experimentalset-up appear to have occurred with about 6-10-fold higher efficiencythan the Sleeping Beauty mediated transpositions.

A couple of additional conclusions can be drawn from this transpositionexperiment: First, from the IgG/EGFP double positive cells sorted on daytwo post transfection, about 75% of cells remained stably EGFP+ in thePiggyBac transpositions, as ca. 40% and 30% of the PB-Ac10 and PB-hBU12transposition generate EGFP+ cells lacking IgG surface expression. Thesecells most likely have stably transposed only one of the twotransposable IgH and IgL expression vectors, which does not allow forsurface IgG expression, but sufficient to render the cellsEGFP-positive. This also means that from the ca. 38% originallytransiently transfected cells, at least 5% are stably transposed with atleast one transposable Ig expression vector.

The numbers of stably transposed cells for the Sleeping Beautytransposition were lower, than those of the PiggyBac transposition, andafter a first round of FACS sorting of IgH+IgL+transposaseco-transfected cells, only about 5% of stably IgG expressing cells wasobtained. However, if also the stably EGFP positive cells areconsidered, about 9% of stably transposed cells were obtained after thefirst FACS sorting cycle using Sleeping Beauty transposase.

When these stably IgG-positive and IgH/IgL transposed cells were FACSsorted again, over 99% stably IgG expressing cells were obtained (FIG.12, rightmost column), and the stable expression phenotype wasmaintained for over four weeks, without any change in the percentage ofIgG+ cells (data not shown). Therefore, it is concluded that thetransposable expression vectors for human IgH and IgL chains, asdisclosed in this invention, are functional and can stably be integratedinto a mammalian host cell genome with high efficiency.

Example 8: Enrichment of Stably IgG Transposed and IgG Expressing CellsVia Specific Antigen Binding

In order to demonstrate that human IgG expressing proB cells, generatedby transposition of IgH and IgL expression vectors, as disclosed inExample 7 above, can be used for the isolation of antigen-specificcells, decreasing numbers of the proB cell line 63-12, expressinganti-CD30 mAb (see d16, 2× sorted, 63-12+PB-Ac10, of FIG. 12) were mixedwith proB cell line 63-12 expressing anti-CD19 mAb (see d16, 2× sorted,63-12+PB-hBU12, of FIG. 12) at ratios 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶ (seeFIG. 13). A total of 10⁷ cells were stained in 1 ml PBS, supplementedwith 2% FCS, for 30 min on ice, with the following reagents:

-   -   0.1 μg 6×His-tagged, recombinant human CD30 (Sino Biological        Inc., Beijing, China, order #10777-H08H), and    -   10 μl mouse anti-human Ig-kappaLC-APC labeled antibody (Life        Technologies, Invitrogen, order #MH10515).

After these primary reagents were removed from the cells, bycentrifugation and washing in PBS, 2% FCS, a secondary staining wasperformed in 1 ml PBS, supplemented with 2% FCS, for 30 min on ice,with:

-   -   0.1 μg biotinylated anti-His-tag antibody (IBA Life Sciences,        Göttingen, Germany, order #2-1590-001)    -   After this secondary reagent was removed from the cells, by        centrifugation and washing in PBS, 2% FCS, the        CD30-6×His/anti-His-tag-bio combination was detected by staining        in 1 ml PBS, 2% FCS, for 30 min on ice, with:    -   1/500 diluted streptavidin-Phycoerythrin (strep-PE) (Affymetrix        ebioscience, order #12-4317-87) reagent.

After the final FACS staining, the cells were again washed twice inice-cold PBS, 2% FCS, and then resuspended in 1.0 ml PBS, 2% FCS, afterwhich the cells were subjected to FACS analysis and cell sorting ofIg-kappaLC/CD30 positive cells (see FIG. 13).

As can be seen from the results disclosed in FIG. 13, a specificpopulation of IgG positive and anti-CD30 reactive cells is detectable inthe upper-right quadrant of the FACS-plots of the positive control, andas expected the intensity of the FACS signal for surface IgG (detectedvia anti-Ig-kappa-APC) correlates with the FACS signal for anti-CD30resulting in a diagonal staining pattern for this population.

In the mixtures of anti-CD30 mAb expressing cells with anti-CD19 mAbexpressing cells, the level of dilution of the specific anti-CD30 mAbexpressing cells is very well reflected by the frequency of CD30 antigenspecific cells in the upper right quadrant and the stringently definedFACS sorting gate (black square in the upper right quadrant). The veryrare events corresponding to CD30-reactive/IgG positive cells uponincreased dilution of the specific cells (1:10,000, 1:100,000,1:1,000,000) are hardly visible on the printouts of the FACS-dot-plots,even, if increasing numbers of events were acquired, as indicated abovethe individual dot plots. However, the frequency of CD30 detectablecells correlated well with their frequency as expected from the dilutionfactor. From this result it is concluded that the display andantigen-specific detection of cells expressing an antigen-specificantibody by means of transposition mediated human IgG expression on thesurface of mammalian and proB cells, as shown here, can reliably beperformed.

The bottom row of FACS dot-plots in FIG. 13 shows the re-analysis of theFACS sorted cells from the different spike-in dilution experiments. Ascan be seen from the results, the re-analysis of the cells FACS sortedfrom the 1:100, 1:1,000 and 1:10,000 dilution resulted in almost thesame cell population being enriched, which showing ca. 90-95% antigenreactive cells. The FACS sorted cells from the 1:100,000 dilutioncontained a small, additional population that did not fall into the gateof IgG-positive/CD30 reactive cells, but also in this experiment ca. 85%of the FACS sorted cells were antigen-specific IgG-expressing cells.Surprisingly, the highest purity of cells, with regard toCD30-reactivity and IgG expression, resulted from the FACS sort, inwhich only 1 in 1,000,000 had been CD30 antigen specific, and where onlyca. 14 cells had been sorted. This can only be explained that her almostclonal effects need to be considered such that the sort was not amixture from IgG-positive-CD30 reactive cells, but rather a few clonesthat all represented IgG-positive-CD30 reactive cell clones.

Nevertheless, the results of the specific antigen-mediated staining andidentification of antigen-specific antibody expressing cells and theirsuccessful enrichment by preparative FACS-mediated cell sorting clearlydemonstrates the feasibility of the method disclosed herein for theisolation of cells expressing antibodies with a desired bindingphenotype.

Example 9: Instruction for the Generation and Use of Transposable IgHExpression Vectors that can be Used to Switch from Membrane Bound toSecreted Antibody Expression

The transposable Ig expression vectors disclosed in Examples 1 to 4 onlyallow expression of human IgG on the surface of mammalian cells, suchthat the binding phenotype of antibodies can readily be identified andenriched for by antigen-binding to the cells, by means of FACS, asexemplified in Example 8, or by cell-panning or batch enrichment methods(e.g. magnetic bead activated cell sorting, MACS). However, it is oftendesired to rapidly analyze the antigen-binding properties of a givenantibody displayed by a cell also as a secreted antibody in solution.While it is possible to PCR-amplify the relevant V_(H) and V_(L) codingregions of an antigen-specific cell clone into expression vectors forsecreted IgG expression, this approach is time consuming and labourintensive.

In the detailed description of the invention it is already disclosedthat transposable IgH expression constructs can be employed that exploitthe natural “switch” from membrane bound to secreted antibodyexpression, based on alternative splicing of genomic IgH chainconstructs.

This switch from membrane bound to secreted antibody expression can beachieved as follows:

Instead of a cDNA-based expression cassette for human Ig-gamma1 heavychains, the original genomic organization of human Ig-gamma1 gene locusneeds to be cloned into the IgH expression vectors as disclosed beforein Example 4. The sequence of the entire immunoglobulin gene locus ingermline configuration can be retrieved from contig NT_010168 of thehuman genome project, which covers the human Ig heavy chain locuslocated on chromosome 14. The human Ig-gamma1 heavy chain gene locusstarting from the first amino acid of the C_(H)1 domain at the 5′ end to500 bp downstream of the last stop codon of the second membrane-spanningexon gamma1-M2 at the 3′ end has a length of 5807 base pairs anddisplays no internal NheI or BstBI sites. Therefore, this gene locus canbe synthesized with flanking NheI and BstBI sites, that can be used fordirectional cloning. Such a gene synthesized fragment can then directlybe used to replace the cDNA coding region of the membrane-boundgamma1-constant coding region in pPB-EGFP-HC-Ac10 (SEQ ID NO:32)

The DNA sequence of a genomic human Ig-gamma1 fragment to be synthesizedis provided in SEQ ID NO:40 below, at the end of this Example (5′-NheIand 3′-BstBI sites are highlights in boldface print).

The organization of the exon and introns of the human Ig-gamma1-heavychain germline locus, including their membrane spanning exons M1 and M2is depicted in FIG. 14. The coding and non-coding regions in thisgenomic gene fragment left in its original genomic configuration aresupposed to contain all required cis-regulatory elements to allowalternative splicing of an Ig-gamma1 mRNA depending on thedifferentiation stage of the B-lineage cells, in which the mRNA isprocessed (Peterson et al. (2002) Mol. Cell. Biol. 22, 5606-5615). Thecloning of fragment SEQ ID NO:40 into a transposable Ig-gamma1 HCexpression vector can be performed by replacing the C-gamma1 codingregion in pPB-EGFP-HC-Ac10 by digesting pPB-EGFP-HC-Ac10 with NheI andBstBI restriction enzymes and ligating the genomic fragment of SEQ IDNO:40 as a NheI-BstBI digested fragment into the NheI-BstBI linearizedvector fragment of pPB-EGFP-HC-Ac10.

The result of this ligation is shown schematically in FIG. 14, and thesequence of the construct is provided in SEQ ID NO:41 below at the endof this Example.

A-MuLV transformed proB cells, like 63-12 cells, represent a suitablecell line to exploit the natural mechanism of alternative splicing of agenomic Ig-gamma1 HC construct, as it is possible to effect phenotypicdifferentiation of these cells to more mature B-lineage cells, if thetransforming activity of the Abl-kinase is inhibited. This canspecifically be achieved with the Abl-kinase inhibitor Gleevec (alsoknown as Imatinib, or STI-571) (Muljo and Schlissel (2003) NatureImmunol. 4, 31-37). However, if A-MuLV transformed proB cells aretreated with Gleevec, they not only initiate phenotypic differentiationto more mature B lineage stages, but this process is also associatedwith an induction of apoptosis (unpublished observation). This can beprevented by first establishing a 63-12 A-MuLV transformed cell linethat is stably transfected with a bcl-2 expression vector.

The mouse bcl-2 mRNA sequence can be found in NCBI-Genbank entryNM_009741, and has the following sequence SEQ ID NO:42, shown below atthe end of this Example. This open reading frame translates into thefollowing amino acid sequence SEQ ID NO:43, also provided below, at theend of the Example.

In order to generate a mammalian bcl-2 expression vector, the murinebcl-2 coding region can be gene synthesized with flanking KpnI and XhoIrestriction enzyme sites that are not present in the coding region ofbcl-2 and a KpnI-XhoI double digested gene-synthesized DNA fragment canbe ligated into pCDNA3.1-hygro(+) described further above in order togenerate a mammalian expression vector for bcl-2 that can stably betransfected into 63-12 cells in order to select for stable bcl-2transfectants.

The entire sequence of the pCDNA3.1-hygro(+) expression vectorcontaining the murine bcl-2 gene inserted into the KpnI and XhoIrestriction sites of the multiple cloning site is provided as SEQ IDNO:44 below, at the end of this Example.

In order to facilitate the generation of stable transfectants, thisvector can e.g. be linearized outside of the expression cassettes forbcl-2 or hygromycinB using the enzyme FspI, that linearizes the vectorin the bacterial ampicillin resistance gene. 20 μg of such a linearizedvector can be transfected into 2×10⁶ 63-12 cells by electroporation at950 μF/300V exactly as disclosed further above for the transfection oftransposable vectors. Following electroporation, the cells can then bediluted in 100 ml growth medium and plated into five 96-well plates witheach 200 μl/well, which will result in the plating of ca. 4×10³cells/well.

Stable transfectants can then selected by adding 800 μg/ml hygromycinB48 hours post transfection. Individual stably transfected cell clones,of which 20-100 can be expected from such an experiment, can then beobtained 2-3 weeks later. Stable bcl-2 transfected cell clones are bestfunctionally tested for their ability to protect cells from apoptosis,by measuring the survival of individual clones upon exposure of 0.1 to 1mM Gleevec (Imatinib, or STI-571). Once a 63-12 stable bcl-2transfectant is identified that has high resistance to Gleevec(Imatinib, or STI-571), this clone can be utilized as a host cell forexpression of human IgG from transposable genomic Ig-gamma1 HC andIg-kappa LC expression vectors, e g utilizing vectors SEQ ID NO:41 andSEQ ID NO:36.

These vector can be co-transfected with PiggyBac expression vector (SEQID NO:13) into the stably Bcl-2 transfected 63-12 cells, and stablytransposed and IgG expressing cells can be established as describedfurther above (equivalent to Example 7). Because proB cells represent adifferentiation stage, where endogenous immunoglobulin is expressed asmembrane bound immunoglobulin, it can be expected that also the Ig-HCexpressed from a transposable Ig-gamma1 HC expression vector in genomicconfiguration will be expressed as membrane bound version.

However, if the cells are treated with 0.1 to 1 mM Gleevec (Imatinib, orSTI-571), the Abl-kinase encoded by the A-MuLV is specificallyinhibited, the cells are no longer transformed and continue theirintrinsic differentiation program to more mature B cell differentiationstages. In vitro, this differentiation is independent of functionallyexpressed Ig proteins (Grawunder et al. (1995) Int. Immunol. 7,1915-1925). It has even been shown that in vitro differentiation ofnon-transformed proB cells renders them responsive to T cell derivedanti-IL4 and CD40 stimulation, upon which the cells even differentiateinto plasma cell stage cells undergoing class-switch recombination andwhere they can be fused with myeloma cells to generate hybridomas(Rolink et al. (1996) Immunity 5, 319-330).

This means that also A-MuLV transformed 63-12 cells, which are renderedresistant to apoptosis by stable expression of bcl-2 can bedifferentiated into cells of the plasma cell stage upon treatment withGleevec and simultaneous incubation with 10 μg/ml agonistic anti-CD40antibody, and 100 U/ml recombinant IL4, exactly as described in Rolinket al. (1996) Immunity 5, 319-330.

This treatment will induce a change in the cellular differentiationprogram, that will change the cellular alternative splicing program frommembrane bound IgG expression to secreted IgG expression from an Ig HCexpression construct in genomic organization. This will allow theproduction of secreted antibody from replica plated cell clonesidentified and isolated by surface display and antigen binding, withoutthe need to re-clone V_(H) and V_(L) coding regions from selected cellclones and without the need to ligate them into expression vectors forsecreted IgG antibodies. This is a functional feature of the vectorsthat cannot easily be incorporated in most mammalian cell expressionsystem, particularly not into many virus-based expression systems, inwhich such extended genomic expression vectors cannot easily beinserted.

Sequences Referred to in this Example 9

SEQ ID NO:40 (5812 bp long genomic human Ig-gamma1-heavy chain gene)

SEQ ID NO:41 (transposable Ig-gamma1-HC expression vector in genomicconfiguration)

SEQ ID NO:42 (murine bcl-2 coding region)

SEQ ID NO:43 (amino acid sequence of murine Bcl-2 protein

SEQ ID NO:44 (pCDNA3.1-hygro(+)-bcl2 mammalian expression vector)

Example 10: Instruction for the Generation of Vectors Encoding BasicHuman Antibody Heavy and Light Chain Libraries as PiggyBac TransposableVectors

In order to generate simple transposable DNA libraries encoding humanantibody heavy and light chain libraries, only the V_(L) and V_(H)regions from transposable vectors pIRES-EGFP-T1T2-IgL of Example 2 andpIRES-EGFP-T1T2-IgH of Example 3, respectively, need to be replaced.This can be done by gene synthesizing human V_(H) and V_(L) codingregions flanked by ClaI and Eco47III restriction enzyme sites, and byallowing nucleotide variations in certain HCDR and LCDR positions, asprovided in SEQ ID NO:9, which encodes libraries for variable heavychain domains, and SEQ ID NO:10, which encodes libraries for variablelight chain domains, and which are provided at the end of this Example.Both of these sequences contain a stretch of N-sequences in the HCDR3(boldface), and LCDR3 (boldface), respectively. Both SEQ ID NO:9 and SEQID NO:10 sequences are flanked by ClaI and Eco47III restriction enzymes(underlined), respectively, including four nucleotides flanking therestriction enzyme sites (highlighted in lowercase letters at the endsof the sequence), allowing proper restriction enzyme digestion of thegene-synthesized DNA fragments and directed ligation into ClaI-Eco47IIIlinearized pIRES-EGFP-T1T2-IgH and pIRES-EGFP-T1T2-IgL backbones,respectively.

This way, diverse transposable DNA libraries, encoding antibody heavyand light chains on separate vectors, in which the expression of theantibody chains are transcriptionally and therefore operably linked to agreen fluorescent marker protein can be generated.

SEQ ID NO:9 (VL domain coding region with variable N-sequence variationat positions encoding LCDR3)

SEQ ID NO:10 (V_(H) domain coding region with variable N-sequencevariation at positions encoding HCDR3)

Example 11: Instructions for the Generation of a Basic Sleeping BeautyTransposable Human Ig-Kappa Light Chain Expression Library

In order to generate a diverse Sleeping Beauty transposable DNA libraryencoding human antibody light chain libraries, the V_(L) region ofSleeping Beauty transposable vector pIRES-EGFP-sbT1T2-IgL of Example 5needs to be replaced with a diverse V_(L) gene repertoire. This can bedone by gene synthesizing of human V_(L) coding regions flanked by ClaIand Eco47III restriction enzyme sites, and by allowing nucleotidevariations in certain HCDR and LCDR positions, as already provided inSEQ ID NO:10 above. The SEQ ID NO:10 sequence is flanked by ClaI andEco47III restriction enzymes allowing directed ligation intoClaI-Eco47III linearized pIRES-EGFP-sbT1T2-IgL. This way a SleepingBeauty transposable DNA library encoding diverse human antibody lightchain can be generated.

This way, diverse transposable DNA libraries, encoding antibody heavyand light chains on separate vectors, in which the expression of theantibody chains are transcriptionally and therefore operably linked to agreen fluorescent marker protein can be generated.

Example 12: Cloning of a Transposable IgL Chain Expression Library

A V-kappa light chain library with randomized LCDR3 region wasconstructed as described below. Six amino acid residues were randomized,i.e. encoded by the codon NNK (N=any nucleotide; K=T or G), whichaccommodates each of the 20 amino acids. The library was based ongermline human Vkappa1-5 and Jkappa2 gene segments and was randomizedbetween the conserved cysteine at the end of the framework 3 region andthe Jkappa2-based framework 4 region as follows: Gln-Gln-(NNK)₆-Thr. Thesequence and overall design of the kappa light chain library is shown inFIG. 15.

A linear DNA molecule encoding the kappa light chain library wasgenerated by PCR. For this, two templates were generated by total genesynthesis (performed by GenScript, Piscataway, N.J., USA). On one hand,a synthetic construct was generated comprising the Vkappa1-5 genesegment cloned into the EcoRV site of pUC57 (Genscript order #SD1176),pUC57_Vkappa1-5 (SEQ ID NO:45); on the other hand, a synthetic constructwas generated comprising the Jkappa2 gene segment fused to the Ckappacoding region cloned into the EcoRV site of pUC57, pUC57_Jkappa2-Ckappa(SEQ ID NO:46).

A first linear DNA comprising the Vkappa1-5 gene segment was PCRamplified from pUC57_Vkappa1-5 using the primers pUC57-1 (5′-CGT TGT AAAACG ACG GCC AG-3′) and LCDR3-B (5′-CTG TTG GCA GTA ATA AGT TGC-3′). Asecond linear DNA comprising the randomized CDR3 region(Gln-Gln-(NNK)₆-Thr), the Jkappa2 gene segment and the Ckappa constantregion was amplified from pUC57_Jkappa2-Ckappa using the primersLCDR3-NNK6-F (5′-GCA ACT TAT TAC TGC CAA CAG NNK NNK NNK NNK NNK NNK ACTTTT GGC CAG GGG ACC AAG-3′) and pUC57-2 (5′-TCA CAC AGG AAA CAG CTATG-3′). To prevent introduction of a sequence bias due to priming of therandomized region of the primer LCDR3-NNK6-F on pUC57_Jkappa2-Ckappa,the plasmid was first linearized by digestion with the restrictionenzyme ScaI (FIG. 17A).

The resulting DNA molecules (SEQ ID NO:47 and SEQ ID NO:48) displayed anoverlap of 21 bp and were assembled by PCR overlap extension using theprimers pUC57-1 and pUC57-2, generating a DNA molecule comprising thekappa light chain library flanked by Nod and AsuII (=BstBI) restrictionsites as shown in FIG. 15. The PCR amplicon of the V-kappa light chainlibrary was subjected to PCR-fragment sequencing, and the result shownin FIG. 18, demonstrate that indeed the expected diversity wasintroduced as designed in the positions of the LCDR3, as evidenced byoverlapping electropherogram signals in the randomized positions. ThisPCR fragment was digested with the restriction endonucleases NotI andBstBI (an isoschizomer of AsuII) and cloned into thePiggyBac-transposable vector pPB-EGFP_HC-g1 (SEQ ID NO:049, resulting ina library consisting of 5.2×10⁷ independent clones. The size of thislibrary can easily be increased by a factor 10 by scaling up theligation reaction.

Light chain libraries incorporating distinct randomization designs, orcomprising Vkappa and Jkappa gene segments other than the ones used inthis example, can be produced the same way. Likewise, the strategydescribed here can be employed for the production of Vlambda light chainlibraries.

Sequences Referred to in this Example 12

SEQ ID NO:45 (pUC57_Vkappa1-5)

SEQ ID NO:46 (pUC57_Jkappa2-C-kappa)

SEQ ID NO:47 (Vkappa1-5 PCR product)

SEQ ID NO:48 (NNK6-Jkappa2-C-kappa PCR product

Example 13: Cloning of Transposable IgH Chain Expression Libraries withVariable HCDR3 Length

A human gamma1 heavy chain library with randomized HCDR3 region wasconstructed as described below. Several amino acid residues wererandomized, i.e. encoded by the codon NNK (N=any nucleotide; K=T or G),which accommodates each of the 20 amino acids. The library was based onthe V_(H)3-30 and J_(H)4 gene segments and was randomized between theconserved Cysteine residue at the end of the framework 3 region and theJ_(H)4-based framework 4 region as follows:Ala-Lys/Arg-(NNK)_(n)-Asp-NNK. Various HCDR3 lengths were explored, withn=4, 6, 8, or 10 (NNK4, NNK6, NNK8, and NNK10 randomization). Thesequence and overall design of the gamma heavy chain library is shown inFIG. 16.

A linear DNA molecule encoding the heavy chain variable region (V_(H))library was generated by PCR. For this, two templates were generated bytotal gene synthesis (performed by GenScript, Piscataway, N.J., USA). Onone hand, a synthetic construct was generated comprising the V_(H)3-30gene segment cloned into the EcoRV site of pUC57, pUC57_V_(H)3-30 (SEQID NO:49); on the other hand, a synthetic construct was generatedcomprising the J_(H)4 gene segment cloned into the EcoRV site of pUC57,pUC57_J_(H)4 (SEQ ID NO:50).

A first linear DNA comprising the V_(H)3-30 gene segment was PCRamplified from pUC57_V_(H)3-30 using the primers V_(H)3-30-F (5′-GAT ATCCAA TGC GGC CGC ATG-3′) and HCDR3-B (5′-CGC ACA GTA ATA CAC AGC CGTG-3′). Additional linear DNA molecules comprising the randomized HCDR3regions Ala-Lys/Arg-(NNK)₄-Asp-NNK, Ala-Lys/Arg-(NNK)₆-Asp-NNK,Ala-Lys/Arg-(NNK)₈-Asp-NNK, or Ala-Lys/Arg-(NNK)₁₀-Asp-NNK fused to theJ_(H)4 gene segment were amplified from pUC57_JH4 using, respectively,the primers HCDR3-NNK4-F (5′-CAC GGC TGT GTA TTA CTG TGC GAR GNN KNN KNNKNN KGA CNN KTG GGG CCA AGG AAC CCT GGT C-3′), HCDR3-NNK6-F (5′-CAC GGCTGT GTA TTA CTG TGC GAR GNN KNN KNN KNN KNN KNN KGA CNN KTG GGG CCA AGGAAC CCT GGT C-3′), HCDR3-NNK8-F (5′-CAC GGC TGT GTA TTA CTG TGC GAR GNNKNN KNN KNN KNN KNN KNN KNN KGA CNN KTG GGG CCA AGG AAC CCT GGT C-3′),or HDR3-NNK10-F (5′-CAC GGC TGT GTA TTA CTG TGC GAR GNN KNN KNN KNN KNNKNN KNN KNN KNN KNN KGA CNN KTG GGG CCA AGG AAC CCT GGT C-3′) incombination with the primer pUC57-3 (5′-CAG GTT TCC CGA CTG GAA AG-3′).To prevent introduction of a sequence bias due to priming of therandomized region of the primers HCDR3-NNK4-F, HCDR3-NNK6-F,HCDR3-NNK8-F and HCDR3-NNK10-F on pUC57_J_(H)4, the plasmid was firstlinearized by digestion with the restriction enzyme DrdI (FIG. 17B).

The resulting V_(H)3-30 PCR product (SEQ ID NO:51) displayed an overlapof 22 bp with the NNK4-J_(H)4, NNK6-J_(H)4, NNK8-J_(H)4 and NNK10-J_(H)4PCR products (SEQ ID NO:52 to 55), and was assembled with each by PCRoverlap extension in 4 separate reactions, using the primers V_(H)3-30-Fand pUC57-3. The resulting DNA molecules comprised the V_(H) libraryflanked by NotI and NheI restriction sites as shown in FIG. 16. All PCRamplicons obtained from the PCRs employing the NNK4-J_(H)4, NNK6-J_(H)4,NNK8-J_(H)4 and NNK10-J_(H)4 degenerate oligos were subjected to directDNA sequencing, and it was confirmed that the designed randomization ofthe HCDR3 positions was obtained, as expected. This is shown by way ofexample in FIG. 18 (B), where the electropherogram of the regionspanning the HCDR3 is provided. The randomized positions show expectedsequence peak overlays demonstrating the nucleotide diversity in thesepositions (FIG. 18). The 4 different V_(H) library DNAs were mixed inequimolar ratio, digested with the restriction endonucleases Nod andNheI and cloned into the PiggyBac-transposable vector pPB-EGFP_HC-gamma1(SEQ ID NO:22), upstream of the gamma1 heavy chain constant region,resulting in a library consisting of 3.7×10⁷ independent clones. Thesize of this library can easily be increased by a factor 10 by scalingup the ligation reaction.

Heavy chain libraries incorporating distinct randomization designs, orcomprising V_(H) and J_(H) gene segments other than the ones used inthis example, can be produced the same way.

DNA Sequences Referred to in this Example 13

SEQ ID NO:49 (pUC57_VH3-30)

SEQ ID NO:50 (pUC57_J_(H)4)

SEQ ID NO:51 (V_(H)3-30 PCR product)

SEQ ID NO:52 (NNK4-J_(H)4 PCR product)

SEQ ID NO:53 (NNK6-J_(H)4 PCR product)

SEQ ID NO:54 (NNK8-J_(H)4 PCR product)

SEQ ID NO:55 (NNK10-J_(H)4 PCR product)

Example 14: Identification of Variable Light and Heavy Chain CodingRegions from Antigen-Reactive, Enriched and Stably Transposed Host Cells

Due to the stable integration of the transposable expression vectorsencoding antibody heavy and light chains in the host, the variable heavyand light chain coding regions can be re-isolated in a straightforwardway by standard PCR amplification followed by direct sequencing of thePCR amplicons or, upon re-cloning, from re-cloned plasmid vectors. Forthis, isolated cells or cell clones, expressing antigen-specificantibodies are centrifuged for 5 minutes at 1200×g. Total RNA isisolated from these cells using TRIzol reagent (Sigma-Aldrich). Firststrand cDNA can be synthesized with PowerScript (Clontech-LifeTechnologies) using an oligio-dT primer. The light chain coding regionscan then amplified by PCR using the primers SP-F (5′-GAG GAG GAG GCG GCCGCC ATG AAT TTT GGA C-3′) and CK-rev (5′-GAG GAG GAG TTC GAA AGC GCT AACACT CTC-3′), which will result in a PCR amplicon of ca. 740 bp,depending on length of the V-kappa region contained in the PCR amplicon.If desired, this PCR amplicon can be digested with restrictionendonucleases NotI and BstBI, and cloned into the vector pPB-EGFP_HC-g1(SEQ ID NO:22), in order to subclone individual clones of the PCRamplicon for further sequence identification. Individual V-kappa regionclones can then be subjected to sequencing using the primer pPB-seq13(5′-GGC CAG CTT GGC ACT TGA TG-3′), binding in the EF1-alpha promoter,upstream of the cloned V-coding region.

The heavy chain variable regions can be PCR amplified on cDNA, generatedas above, using the primers SP-F (5′-GAG GAG GAG GCG GCC GCC ATG AAT TTTGGA C-3′) and CG-revseq-1 (5′-GTT CGG GGA AGT AGT CCT TG-3′) that willresult in a PCR amplicon of ca. 530 bp expected size, depending in thelength of the V_(H)-region contained in the PCR amplicon. If desired,this PCR amplicon can be digested with restriction endonucleases NotIand NheI, and cloned into the vector pPB-EGFP_HC-g1 (SEQ ID NO:22).Individual clones are then subjected to sequencing of the V_(H)-regionusing the primer pPB-seq13 (5′-GGC CAG CTT GGC ACT TGA TG-3′), bindingin the EF1-alpha promoter, upstream of the cloned V-coding region.

EXEMPLARY EMBODIMENTS OF THE INVENTION

1. A method for identifying a polypeptide having a desired bindingspecificity or functionality, comprising:

(i) generating a diverse collection of polynucleotides encodingpolypeptides having different binding specificities or functionalities,wherein said polynucleotides comprise a sequence coding for apolypeptide disposed between first and second inverted terminal repeatsequences that are recognized by and functional with a least onetransposase enzyme;(ii) introducing the diverse collection of polynucleotides of (i) intohost cells;(iii) expressing at least one transposase enzyme functional with saidinverted terminal repeat sequences in said host cells so that saiddiverse collection of polynucleotides is integrated into the host cellgenome to provide a host cell population that expresses said diversecollection of polynucleotides encoding polypeptides having differentbinding specificities or functionalities;(iv) screening said host cells to identify a host cell expressing apolypeptide having a desired binding specificity or functionality; and(v) isolating the polynucleotide sequence encoding said polypeptide fromsaid host cell.

Embodiment A2

A method according to Embodiment A1, wherein said polynucleotides areDNA molecules.

Embodiment A3

A method according to Embodiment A1, wherein said polynucleotidescomprise a ligand-binding sequence of a receptor or a target-bindingsequence of a binding molecule.

Embodiment A4

A method according to Embodiment A1, wherein said polynucleotidescomprise an antigen-binding sequence of an antibody.

Embodiment A5

A method according to Embodiment A1, wherein said polynucleotidescomprise a sequence encoding a VH or VL region of an antibody, or anantigen-binding fragment thereof.

Embodiment A6

A method according to Embodiment A1, wherein said polynucleotidescomprise a sequence encoding an antibody VH region and an antibody VLregion.

Embodiment A7

A method according to Embodiment A1, wherein said polynucleotidescomprise a sequence encoding a full-length immunoglobulin heavy chain orlight chain, or an antigen-binding fragment thereof.

Embodiment A8

A method according to Embodiment A1, wherein said polynucleotidescomprise a sequence encoding a single-chain Fv or a Fab domain.

Embodiment A9

A method according to Embodiment A1, wherein generating said diversecollection of polynucleotides comprises subjecting V region genesequences to PCR under mutagenizing conditions.

Embodiment A10

A method according to Embodiment A1, wherein generating said diversecollection of polynucleotides comprises gene synthesis.

Embodiment A11

A method according to Embodiment A1, wherein generating said diversecollection of polynucleotides comprises PCR amplification of V regionrepertoires from vertebrate B cells.

Embodiment A12

A method according to Embodiment A1, wherein said diverse collection ofpolynucleotides comprises plasmid vectors.

Embodiment A13

A method according to Embodiment A1, wherein said diverse collection ofpolynucleotides comprises double-stranded DNA PCR amplicons.

Embodiment A14

A method according to Embodiment A4, wherein said antigen-bindingsequence is of a vertebrate.

Embodiment A15

A method according to Embodiment A4, wherein said antigen-bindingsequence is mammalian.

Embodiment A16

A method according to Embodiment A4, wherein said antigen-bindingsequence is human.

Embodiment A17

A method according to Embodiment A12, wherein said plasmid vectorsfurther encode a marker gene.

Embodiment A18

A method according to Embodiment A17, wherein said marker is selectedfrom the group consisting of: a fluorescent marker, a cell surfacemarker and a selectable marker.

Embodiment A19

A method according to Embodiment A17, wherein said marker gene sequenceis upstream or downstream of the sequence encoding the polypeptidehaving a binding specificity or functionality, but between the invertedterminal repeat sequences.

Embodiment A20

A method according to Embodiment A17, wherein said marker gene sequenceis downstream of said sequence encoding a polypeptide having bindingspecificity or functionality and separated by an internal ribosomalentry site.

Embodiment A21

A method according to Embodiment A1, wherein step (ii) comprisesintroducing into said host cells polynucleotides comprising sequencesencoding immunoglobulin VH or VL regions, or antigen-binding fragmentsthereof, and wherein said VH and VL region sequences are encoded onseparate vectors.

Embodiment A22

A method according to Embodiment A21, wherein step (ii) comprisesintroducing into said host cells polynucleotides comprising sequencesencoding full-length immunoglobulin heavy or light chains, orantigen-binding fragments thereof, wherein said full-length heavy andlight chain sequences are on separate vectors.

Embodiment A23

A method according to Embodiment A21, wherein said vectors comprising VHsequences and said vectors comprising VL sequences are introduced intosaid host cells simultaneously.

Embodiment A24

A method according to Embodiment A21, wherein said vectors comprising VHsequences and said vectors comprising VL sequences are introduced intosaid host cells sequentially.

Embodiment A25

A method according to Embodiment A1, wherein step (ii) comprisesintroducing into said host cells a vector comprising sequences encodingantibody VH and VL chains.

Embodiment A26

A method according to Embodiment A1, wherein step (ii) comprisesintroducing into said host cells a vector comprising sequences encodinga full-length immunoglobulin heavy chain and a full-lengthimmunoglobulin light chain.

Embodiment A27

A method according to Embodiment A21, wherein said vector comprising theVH sequence comprises inverted terminal repeat sequences that arerecognized by a different transposase enzyme than the inverted terminalrepeat sequences in the vector comprising the VL sequence.

Embodiment A28

A method according to Embodiment A1, wherein the host cells of step (ii)are vertebrate cells.

Embodiment A29

A method according to Embodiment A28, wherein said host cells aremammalian.

Embodiment A30

A method according to Embodiment A29, wherein said host cells are humanor rodent cells.

Embodiment A31

A method according to Embodiment A28, wherein said vertebrate host cellsare lymphoid cells.

Embodiment A32

A method according to Embodiment A31, wherein said host cells are Bcells.

Embodiment A33

A method according to Embodiment A32, wherein said host cells areprogenitor B cells or precursor B cells.

Embodiment A34

A method according to Embodiment A33, wherein said host cells areselected from the group consisting of: Abelson-Murine Leukemia virustransformed progenitor B cells or precursor B cells and early,immunoglobulin-null EBV transformed human proB and preB cells.

Embodiment A35

A method according to Embodiment A32, wherein said host cells areselected from the group consisting of: Sp2/0 cells, NSO cells, X63cells, and Ag8653 cells.

Embodiment A36

A method according to Embodiment A29, wherein said host cells areselected from the group consisting of: CHO cells, Per.C6 cells, BHKcells, and 293 cells.

Embodiment A37

A method according to Embodiment A1, wherein said expressing step (iii)comprises introducing into said host cells an expression vector encodinga transposase enzyme that recognizes and is functional with an least oneinverted terminal repeat sequence.

Embodiment A38

A method according to Embodiment A37, wherein said vector encoding saidtransposase enzyme is introduced into said host cells concurrently withor prior or subsequent to the diverse collection of polynucleotides.

Embodiment A39

A method according to Embodiment A37, wherein said transposase enzyme istransiently expressed in said host cell.

Embodiment A40

A method according to Embodiment A1, wherein said expressing step (iii)comprises inducing an inducible expression system that is stablyintegrated into the host cell genome.

Embodiment A41

A method according to Embodiment A40, wherein said inducible expressionsystem is tetracycline-inducible or tamoxifen-inducible.

Embodiment A42

A method according to Embodiment A1, wherein said screening step (iv)comprises magnetic activated cell sorting (MACS), fluorescence activatedcell sorting (FACS), panning against molecules immobilized on a solidsurface panning, selection for binding to cell-membrane associatedmolecules incorporated into a cellular, natural or artificiallyreconstituted lipid bilayer membrane, or high-throughput screening ofindividual cell clones in multi-well format for a desired functional orbinding phenotype.

Embodiment A43

A method according to Embodiment A1, wherein said screening step (iv)comprises screening to identify polypeptides having a desiredtarget-binding specificity or functionality.

Embodiment A44

A method according to Embodiment A1, wherein said screening step (iv)comprises screening to identify antigen-binding molecules having adesired antigen specificity.

Embodiment A45

A method according to Embodiment A44, wherein said screening stepfurther comprises screening to identify antigen-binding molecules havingone or more desired functional properties.

Embodiment A46

A method according to Embodiment A1, wherein said screening step (iv)comprises multiple cell enrichment cycles with host cell expansionbetween individual cell enrichment cycles.

Embodiment A47

A method according to Embodiment A1, wherein said step (v) of isolatingthe polynucleotide sequence encoding the polypeptide having a desiredbinding specificity or functionality comprises genomic or RT-PCRamplification or next-generation deep sequencing.

Embodiment A48

A method according to Embodiment A1, further comprising (vi) affinityoptimizing the polynucleotide sequence obtained in (v).

Embodiment A49

A method according to Embodiment A48, wherein said affinity optimizationcomprises genomic PCR or RT-PCR under mutagenizing conditions.

Embodiment A50

A method according to Embodiment A49, further comprising subjecting themutagenized sequences to steps (i)-(v) of claim 1.

Embodiment A51

A method according to Embodiment A1, wherein said inverted terminalrepeat sequences are from the PiggyBac transposon system.

Embodiment A52

A method according to Embodiment A51, wherein the sequence encoding theupstream PiggyBac inverted terminal repeat sequence comprises SEQ IDNO:1.

Embodiment A53

A method according to Embodiment A51, wherein the sequence encoding thedownstream PiggyBac inverted terminal repeat sequence comprises SEQ IDNO:2.

Embodiment A54

A method according to Embodiment A5, wherein said VH or VL regionsequences encode a sequence derived from a human anti-TNF alphaantibody.

Embodiment A55

A method according to Embodiment A54, wherein said human anti-TNF alphaantibody is D2E7.

Embodiment A56

A method according to Embodiment A55, wherein the VH and VL regions ofD2E7 are encoded by separate transposable vectors.

Embodiment A57

A method according to Embodiment A56, wherein said vector comprisingsaid VL region sequence comprises SEQ ID NO:5.

Embodiment A58

A method according to Embodiment A56, wherein said vector comprisingsaid VH region sequence comprises SEQ ID NO:8.

Embodiment A59

A method according to Embodiment A56, wherein said vector comprisingsaid VH region sequence comprises a randomized sequence as set forth inSEQ ID NO:9.

Embodiment A60

A method according to Embodiment A56, wherein said vector comprisingsaid VL region sequence comprises a randomized sequence as set forth inSEQ ID NO:10.

Embodiment A61

A method according to Embodiment A1, wherein step (iii) comprisesintroducing into said host cell a vector comprising a sequence encodinga functional PiggyBac transposase.

Embodiment A62

A method according to Embodiment A61, wherein said vector comprises SEQID NO:11.

Embodiment A63

A method according to Embodiment A61, wherein said vector encodes SEQ IDNO:12, or a sequence with at least 95% amino acid sequence homology andhaving the same or similar inverted terminal repeat sequencespecificity.

Embodiment A64

A method according to Embodiment A claim 1, wherein said invertedterminal repeat sequences are recognized by and functional with at leastone transposase selected from the group consisting of: PiggyBac,Sleeping Beauty, Frog Prince, Himar1, Passport, Minos, hAT, Tol1, Tol2,Ac/Ds, PIF, Harbinger, Harbinger3-DR, and Hsmar1.

Embodiment B65

A library of polynucleotide molecules encoding polypeptides havingdifferent binding specificities or functionalities, comprising aplurality of polynucleotide molecules, wherein said polynucleotidemolecules comprise a sequence encoding a polypeptide having a bindingspecificity or functionality disposed between inverted terminal repeatsequences that are recognized by and functional with at least onetransposase enzyme.

Embodiment B66

A library according to Embodiment B65, wherein said polynucleotides areDNA molecules.

Embodiment B67

A library according to Embodiment B65, wherein said polynucleotidescomprise a ligand-binding sequence of a receptor or a target-bindingsequence of a binding molecule.

Embodiment B68

A library according to Embodiment B65, wherein said polynucleotidescomprise at least one sequence encoding an antigen-binding sequence ofan antibody.

Embodiment B69

A library according to Embodiment B65, wherein said polynucleotidescomprise a sequence encoding a VH or VL region of an antibody or anantigen-binding fragment thereof.

Embodiment B70

A library according to Embodiment B65, wherein said polynucleotidescomprise a sequence encoding an antibody VH region and an antibody VLregion.

Embodiment B71

A library according to Embodiment B65, wherein said polynucleotidescomprise a sequence encoding a full-length immunoglobulin heavy chain orlight chain, or an antigen-binding fragment thereof.

Embodiment B72

A library according to Embodiment B65, wherein said polynucleotidescomprise a sequence encoding a single-chain Fv or a Fab domain.

Embodiment B73

A library according to Embodiment B65, wherein said polynucleotidemolecules are plasmids.

Embodiment B74

A library according to Embodiment B65, wherein said polynucleotidemolecules are double stranded DNA PCR amplicons.

Embodiment B75

A library according to Embodiment B73, wherein said plasmids furthercomprise a sequence encoding a marker gene.

Embodiment B76

A library according to Embodiment B73, wherein said plasmids furthercomprise a sequence encoding a transposase enzyme that recognizes and isfunctional with the inverted terminal repeat sequences.

Embodiment C77

A method for generating a library of transposable polynucleotidesencoding polypeptides having different binding specificities orfunctionality, comprising:

-   -   (i) generating a diverse collection of polynucleotides        comprising sequences encoding polypeptides having different        binding specificities or functionalities, wherein said        polynucleotides comprise a sequence encoding polypeptide having        a binding specificity or functionality disposed between inverted        terminal repeat sequences that are recognized by and functional        with a least one transposase enzyme.

Embodiment D78

A vector comprising a sequence encoding a VH or VL region of anantibody, or antigen-binding portion thereof, disposed between invertedterminal repeat sequences that are recognized by and functional with atleast one transposase enzyme.

Embodiment D79

A vector according to Embodiment D78, comprising a sequence encoding afull-length heavy or light chain of an immunoglobulin.

Embodiment D80

A vector according to Embodiment D78, wherein said VH or VL regionsequence is randomized.

Embodiment D81

A vector according to Embodiment D78, wherein said inverted terminalrepeat sequences are recognized by and functional with the PiggyBactransposase.

Embodiment D82

A vector according to Embodiment D78, wherein said VH or VL regionsequence is derived from an anti-TNF alpha antibody.

Embodiment D83

A vector according to Embodiment D82, wherein said antibody is D2E7.

Embodiment D84

A vector according to Embodiment D78, comprising at least one sequenceselected from the group consisting of: SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:17, and SEQ ID NO:19.

Embodiment D85

A host cell comprising a vector according to any one of claims 78-84.

Embodiment D86

A host cell according to Embodiment D85 further comprising an expressionvector comprising a sequence encoding a transposase that recognizes andis functional with at least one inverted terminal repeat sequence in thevector encoding said VH or VL region sequence.

Embodiment E87

An antibody produced by a method comprising claim 1.

Embodiment E88

A method according to Embodiment A1, wherein said inverted terminalrepeat sequences are from the Sleeping Beauty transposon system.

Embodiment E89

A method according to Embodiment A88, wherein the sequence encoding theupstream Sleeping Beauty inverted terminal repeat sequence comprises SEQID NO:14.

Embodiment E90

A method according to Embodiment A88, wherein the sequence encoding thedownstream Sleeping Beauty inverted terminal repeat sequence comprisesSEQ ID NO:15.

Embodiment E91

A method according to Embodiment A88, wherein step (iii) comprisesexpressing in said host cell a vector comprising a functional SleepingBeauty transposase.

Embodiment A92

A method according to Embodiment A48, wherein said polynucleotidesequence obtained in (v) comprises a sequence encoding a VH or VL regionof an antibody, or an antigen-binding fragment thereof, and wherein saidantibody optimization comprises introducing one or more mutations into acomplementarity determining region or framework region of said VH or VL.

Embodiment B93

A library according to Embodiment B71, wherein said full-lengthimmunoglobulin heavy chain comprises the natural intron/exon structureof an antibody heavy chain.

Embodiment B94

A library according to Embodiment B93, wherein said full-lengthimmunoglobulin heavy chain comprises the endogenous membrane anchordomain.

Embodiment F95

A method for generating a population of host cells capable of expressingpolypeptides having different binding specificities or functionalities,comprising:

-   -   (i) generating a diverse collection of polynucleotides        comprising sequences encoding polypeptides having different        binding specificities or functionalities, wherein said        polynucleotides comprise a sequence encoding a polypeptide        having a binding specificity or functionality disposed between        inverted terminal repeat sequences that are recognized by and        functional with a least one transposase enzyme; and    -   (ii) introducing said diverse collection of polynucleotides into        host cells.

Embodiment D96

A vector according to Embodiment D78, wherein said inverted terminalrepeat sequences are recognized by and functional with the SleepingBeauty transposase

Embodiment A97

A method according to Embodiment A91, wherein step (iii) comprisesexpressing in said host cell a vector comprising SEQ ID NO:17.

Embodiment A98

A method according to Embodiment A91, wherein said vector encodes SEQ IDNO:18, or a sequence with at least 95% amino acid sequence homology andhaving the same or similar inverted terminal repeat sequencespecificity.

1-20. (canceled)
 21. A method of obtaining a polypeptide having adesired binding specificity or functionality, comprising: (i) generatinga diverse collection of polynucleotides encoding polypeptides havingdifferent binding specificities or functionalities, wherein saidpolynucleotides each comprise a sequence encoding a polypeptide, saidsequence disposed between first and second inverted terminal repeatsequences that are recognized by and functional with a least onetransposase enzyme; (ii) introducing the diverse collection ofpolynucleotides of (i) into host cells; (iii) expressing at least onetransposase enzyme functional with said inverted terminal repeatsequences in said host cells so that said diverse collection ofpolynucleotides is integrated into the host cell genomes to provide ahost cell population that expresses said polypeptides having differentbinding specificities or functionalities; (iv) screening said host cellsto identify a host cell expressing a polypeptide having a desiredbinding specificity or functionality; and (v) isolating thepolynucleotide sequence encoding said polypeptide identified in step(iv) from said host cell.
 22. The method according to claim 21, whereinsaid polynucleotides comprise a sequence encoding: a) a ligand-bindingdomain of a receptor or a target-binding domain of a binding molecule,b) an antigen-binding domain of an antibody, c) a V_(H) or V_(L) regionof an antibody or an antigen-binding fragment thereof, d) an antibodyV_(H) region and an antibody V_(L) region, e) a full-lengthimmunoglobulin heavy chain or light chain or an antigen-binding fragmentthereof, or f) a single-chain Fv or a Fab domain.
 23. The methodaccording to claim 22, wherein generating said diverse collection ofpolynucleotides comprises subjecting V region gene sequences to PCRunder mutagenizing conditions.
 24. The method according to claim 21,wherein step (ii) comprises introducing into said host cellspolynucleotides comprising sequences encoding: a) immunoglobulin V_(H)or V_(L) regions or antigen-binding fragments thereof, wherein saidV_(H) and V_(L) region sequences are encoded on separate vectors, b)full-length immunoglobulin heavy or light chains, or antigen-bindingfragments thereof, wherein said full-length heavy and light chainsequences are encoded on separate vectors, c) an antibody V_(H) andV_(L) chains encoded on the same vector, or d) a full-lengthimmunoglobulin heavy chain and a full-length immunoglobulin light chainencoded on the same vector.
 25. The method according to claim 21,wherein said expressing step (iii) comprises introducing into said hostcells an expression vector encoding a transposase enzyme that recognizesand is functional with an least one inverted terminal repeat sequence,wherein said transposase enzyme is transiently expressed in said hostcells.
 26. The method according to claim 21, wherein said screening step(iv) comprises: magnetic activated cell sorting (MACS), fluorescenceactivated cell sorting (FACS), panning against molecules immobilized ona solid surface, selection for binding to cell-membrane associatedmolecules incorporated into a cellular, natural or artificiallyreconstituted lipid bilayer membrane, or high-throughput screening ofindividual cell clones in a multi-well format for a desired functionalor binding phenotype.
 27. The method according to claim 21, wherein saidstep (v) of isolating the polynucleotide sequence encoding thepolypeptide having a desired binding specificity or functionalitycomprises genomic or RT-PCR amplification or next-generation deepsequencing.
 28. The method according to claim 21, wherein a) saidinverted terminal repeat sequences are from the PiggyBac transposonsystem or the Sleeping Beauty transposon system, and/or b) step (iii)comprises introducing into said host cells a vector comprising asequence encoding a functional PiggyBac transposase or Sleeping Beautytransposase.
 29. The method according to claim 21, wherein said invertedterminal repeat sequences are recognized by and functional with at leastone transposase selected from the group consisting of: PiggyBac,Sleeping Beauty, Frog Prince, Himar1, Passport, Minos, hAT, Tol1, Tol2,Ac/Ds, PIF, Harbinger, Harbinger3-DR, and Hsmar1. 30-34. (canceled) 35.A vector or set of vectors, comprising a sequence encoding a V_(H) orV_(L) region of an antibody, or antigen-binding portion thereof,disposed between inverted terminal repeat sequences that are recognizedby and functional with at least one transposase enzyme.
 36. The vectoror set of vectors of claim 35, wherein said V_(H) or V_(L) regionsequences encode a sequence derived from a human anti-TNF alphaantibody.
 37. The vector or set of vectors of claim 36, wherein theV_(H) or V_(L) regions are encoded by separate transposable vectors. 38.The vector or set of vectors of claim 36, wherein the antibody sequencesare from D2E7.
 39. The vector or set of vectors of claim 35, wherein afirst vector having the V_(H) sequence comprises inverted terminalrepeat sequences that are recognized by a different transposase enzymethan the inverted terminal repeat sequences in a second vector that hasthe V_(L) sequence.
 40. A host cell comprising a vector or set ofvectors according to claim
 35. 41. The host cell of claim 40, whereinthe host cell is mammalian.
 42. The host cell of claim 41, wherein thehost cell is human or rodent.
 43. The host cell of claim 42, wherein thehost cell is a lymphoid cell.
 44. The host cell of claim 43, wherein thehost cell is selected from: B cell, progenitor B cells, precursor Bcells, Abelson-Murine Leukemia virus transformed progenitor B cells orprecursor B cells, and early, immunoglobulin-null EBV transformed humanproB or preB cells. 45-47. (canceled)
 48. A method for generating apopulation of host cells capable of expressing polypeptides havingdifferent binding specificities or functionalities, comprising: (i)generating a diverse collection of polynucleotides comprising sequencesencoding polypeptides having different binding specificities orfunctionalities, wherein said polynucleotides comprise a sequenceencoding a polypeptide having a binding specificity or functionalitydisposed between inverted terminal repeat sequences that are recognizedby and functional with a least one transposase enzyme; and (ii)introducing said diverse collection of polynucleotides into the host.